Pan Mao

Fabrication and characterization of 20 nm planar nanofluidic channels by glass–glass and glass–silicon bonding

We have characterized glass-glass and glass-Si bonding processes for the fabrication of wide, shallow nanofluidic channels with depths down to the nanometer scale. Nanochannels on glass or Si substrate are formed by reactive ion etching or a wet etching process, and are sealed with another flat substrate either by glass-glass fusion bonding (550 degrees C) or an anodic bonding process. We demonstrate that glass-glass nanofluidic channels as shallow as 25 nm with low aspect ratio of 0.0005 (depth to width) can be achieved with the developed glass-glass bonding technique. We also find that silicon-glass nanofluidic channels, as shallow as 20 nm with aspect ratio of 0.004, can be reliably obtained with the anodic bonding technique. The thickness uniformity of sealed nanofluidic channels is confirmed by cross-sectional SEM analysis after bonding. It is shown that there is no significant change in the depth of the nanofluidic channels due to anodic bonding and glass-glass fusion bonding processes.

Nanofilter array chip for fast gel-free biomolecule separation

We report here a microfabricated nanofilter array chip that can size-fractionate SDS-protein complexes and small DNA molecules based on the Ogston sieving mechanism. Nanofilter arrays with a gap size of 40-180nm were fabricated and characterized. Complete separation of SDS-protein complexes and small DNA molecules were achieved in several minutes with a separation length of 5mm. The fabrication strategy for the nanofilter array chip allows further increasing of the nanofilter density and decreasing of the nanofilter gap size, leading, in principle, to even faster separation.

Artificial molecular sieves and filters: a new paradigm for biomolecule separation

Patterned regular sieves and filters with comparable molecular dimensions hold great promise as an alternative to conventional polymeric gels and fibrous membranes to improve biomolecule separation. Recent developments of microfabricated nanofluidic sieves and filters have demonstrated superior performance for both analytical and preparative separation of various physiologically relevant macromolecules, including proteins. The insights gained from designing these artificial molecular sieves and filters, along with the promising results gathered from their first applications, serve to illustrate the impact that they can have on improving future separation of complex biological samples. Further development of artificial sieves and filters with more elaborate geometrical constraints and tailored surface functionality is believed to provide more promising ideals and results for biomolecule separation, which has great implications for proteomic research and biomarker discovery.

Continuous-Flow Bioseparation Using Microfabricated Anisotropic Nanofluidic Sieving Structures

The anisotropic nanofluidic-filter (nanofilter) array (ANA) is a unique molecular-sieving structure for separating biomolecules. In this protocol we describe the fabrication of planar and vertical ANA chips and how to perform continuous-flow bioseparation using them. This protocol is most useful for bioengineers who are interested in developing automated multistep chip-based bioanalysis systems and assumes previous cleanroom microfabrication knowledge. The ANA consists of a two-dimensional periodic nanofilter array, and the designed structural anisotropy of ANA causes different-sized or charged biomolecules to follow distinct trajectories under applied electric fields, leading to efficient continuous-flow separation. Using microfluidic channels surrounding the ANA, the fractionated biomolecule streams are collected and routed to different fluid channels or reservoirs for convenient sample recovery and downstream bioanalysis. The ANA is physically robust and can be reused repeatedly. Compared with the conventional gel-based separation techniques, ANA offers the potential for faster separation, higher throughput and more convenient sample recovery.

Layer-by-layer deposition of all-nanoparticle multilayers in confined geometries.

Nanofluidic arrays containing high-aspect-ratio nanochannels were used as a platform for the deposition of all nanoparticle multilayers. LbL assembly of 6 nm titania and 15 nm silica nanoparticles resulted in conformal multilayers of uniform thickness throughout the nanochannels. These multilayers are inherently nanoporous with void volume fractions of about 0.5. Compared to unconfined assembly of the same materials on flat substrates, thinner multilayer films were observed for the case of deposition within confined channel geometries because of surface charge-induced electrostatic depletion of the depositing species. Additionally, systematic and reproducible bridging of the nanochannels occurred as multilayer assembly progressed, a phenomenon not seen in our earlier work involving polyelectrolytes. This behavior was attributed to relatively weak nanoparticle adsorption and the resulting formation of large aggregates. These results demonstrate a new route by which confined geometries can be coated and even bridged with a nanoporous multilayer without the need for calcination or other postassembly steps to introduce porosity into the conformal coating.

Rapid Quantification of Disease-Marker Proteins Using Continuous-Flow Immunoseparation in a Nanosieve Fluidic Device

Nanometer-scale fluidic devices offer an alternative to gels for separating biomolecules with better control and accuracy. Here we demonstrate the quantitative analysis of disease-marker proteins by continuously separating the antibody-protein immunocomplexes from the unbound antibodies, utilizing the anisotropically patterned nanosieve array (ANA) structures. The ANA structures, composed of periodically patterned deep channels and shallow regions, allow the small antibodies to pass through the shallow regions easier than the large immunocomplex, when the flow-field is applied in an oblique direction. We examined two proteins used as disease markers, human C-reactive protein (CRP) and human chorionic gonadotropin (hCG), by using fluorescent-labeled polyclonal antibodies. We showed that the size of the immunocomplex and the field strength are the critical factors for the separation, and we successfully demonstrated the quantification of the proteins in the range of 0.05 to 10 microg/mL. Additionally, this device allows a convenient measurement of homogeneous binding kinetics, without the need for repeated binding experiments and immobilizing the molecules. The presented nanofluidic device will be a useful tool for the rapid quantification and the preparative immunoseparation of the target proteins.

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

Nanofluidic molecular filters for efficient protein separation and preconcentration

Regular nanofluidic channels, as thin as 20 nm, have been fabricated and used for protein separation and preconcentration. The nanochannels were fabricated by the substrate-bonding method, and the regularity of the nanochannel has been confirmed by cross-section SEM imaging. A nanofilter array device, with an array of 60-120 nm thin nanofilters, was used for separating small biomolecules (short double-stranded DNA and SDS-coated proteins) based on their size. The sieving of biomolecules (which are comparable to or smaller than the nanofilter gap size) is achieved by steric hindrance of biomolecules within nanochannels. The efficiency of this gel-free separation device is comparable to capillary gel electrophoresis, and can be made better by further optimization. In addition, we have developed a novel biomolecule concentrator using a 40 nm nanofluidic channel as a perm-selective membrane. More than a million times preconcentration of dilute protein or peptide solutions was demonstrated, which was enabled by the stability of the device.