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 ...

Newomics, Inc.

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.

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

Il comportamento di deformazione taglio su scala nanometrica dei due opposti strati aggrecan fine innestato &egrave; stato studiato in soluzioni acquose mediante microscopia a forza atomica e fu osservato a marcatamente dipendono dalla forza ionica del bagno, la presenza di ioni di calcio e il tasso applicato spostamento laterale. Questi risultati forniscono il livello molecolare intuizioni il contributo di aggrecan meccanismi di deformazione di propriet&agrave; materiale a livello di tessuto della cartilagine.

Meccanismi di deformazione delle opposte macromolecole aggrecan cartilagine di taglio su scala nanometrica.

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The nanoscale shear deformation behavior of two opposing end-grafted aggrecan layers was studied in aqueous solutions using atomic force microscopy, and was observed to depend markedly on bath ionic strength, the presence of calcium ions, and the applied lateral displacement rate. These results provide molecular-level insights into the contribution of aggrecan deformation mechanisms to cartilage tissue-level material properties.

Nanoscale shear deformation mechanisms of opposing cartilage aggrecan macromolecules.

Le comportement &#xE0; la d&#xE9;formation de cisaillement &#xE9;chelle nanom&#xE9;trique de deux couches oppos&#xE9;es aggrecane fin greff&#xE9;es a &#xE9;t&#xE9; &#xE9;tudi&#xE9;e dans des solutions aqueuses en utilisant la microscopie &#xE0; force atomique, et a &#xE9;t&#xE9; observ&#xE9;e &#xE0; d&#xE9;pendre fortement sur la force ionique de bain, la pr&#xE9;sence d&#x26;#39;ions calcium, et le taux de d&#xE9;placement appliqu&#xE9; lat&#xE9;rale. Ces r&#xE9;sultats fournissent un aper&#xE7;u au niveau mol&#xE9;culaire dans la contribution des m&#xE9;canismes de d&#xE9;formation aggrecane aux propri&#xE9;t&#xE9;s du cartilage du mat&#xE9;riel tissulaire au niveau.

Nanoscale m&#xE9;canismes de d&#xE9;formation de cisaillement de s&#x26;#39;opposer Macromol&#xE9;cules aggrecane cartilage

Die nanoskaligen Scherung Verformungsverhaltens von zwei entgegengesetzten Ende veredelt Aggrecan Schichten wurde in w&auml;ssrigen L&ouml;sungen mittels Rasterkraftmikroskopie studierte und wurde beobachtet, um Bad Ionenst&auml;rke, die Anwesenheit von Calcium-Ionen und die angewandte Lateralverschiebung-Rate deutlich abh&auml;ngen. Diese Ergebnisse liefern Molekularebene Einblicke in den Beitrag von Aggrecan Verformung Mechanismen zur Knorpel-Gingivaniveau Materialeigenschaften.

Nanoskalige Scheren Verformung Mechanismen der gegen&uuml;berliegenden Knorpel Aggrecan Makromolek&uuml;le.

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El comportamiento nanoescala deformaci&#xF3;n por cizallamiento de dos opuestos injertadas final capas aggrecan se estudi&#xF3; en soluciones acuosas utilizando microscop&#xED;a de fuerza at&#xF3;mica, y se observ&#xF3; que dependen notablemente de la fuerza i&#xF3;nica ba&#xF1;o, la presencia de iones de calcio, y el tipo aplicado desplazamiento lateral. Estos resultados proporcionan a nivel molecular ideas sobre la contribuci&#xF3;n de los mecanismos de deformaci&#xF3;n en las propiedades del cart&#xED;lago aggrecan materiales a nivel de tejidos.

Mecanismos de deformaci&#xF3;n de corte a nanoescala de oponerse a macromol&#xE9;culas del cart&#xED;lago aggrecan

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.

Artificial molecular sieves and filters: a new paradigm for 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.

A Nanofilter Array Chip for Fast Gel-Free Biomolecule Separation.

Many fabrication technologies have been used to build nano/mesoporous materials/filters with a good size control, but the integration of these systems into a microsystem format has been a challenge. Microfabricated nanofilters suffer from small open volume and low-throughput. In this paper, we developed a novel fabrication strategy for generating massively-parallel, regular vertical nanochannel membranes with a uniform, well-controlled gap size of approximately 50 nm and a depth up to approximately 40 microm, by using only standard semiconductor fabrication techniques. The vertical nanofilter membranes were fabricated into an anisotropic nanofilter array, which demonstrates the ability to integrate nanofilters and micron-sized channels/pores seamlessly. We demonstrated efficient continuous-flow separation of large DNAs and small molecules in a two-dimensional vertical nanochannel array device. These ultra-high-aspect-ratio nanochannels have the advantage of large open volume, enabling high-throughput applications.

Massively-parallel ultra-high-aspect-ratio nanochannels as mesoporous membranes.

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.

Rapid quantification of disease-marker proteins using continuous-flow immunoseparation in a nanosieve fluidic device.

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.

Continuous-flow bioseparation using microfabricated anisotropic nanofluidic sieving structures.

In blood vessels with luminal diameter less than 300 µm, red blood cells (RBCs) which are smaller in size and more deformable than leukocytes, migrate to the axial centre of the vessel due to flow velocity gradient within the vessels. This phenomenon displaces the leukocytes to the vessel wall and is aptly termed as margination. Here, we demonstrate using microfluidics that stiffer malaria-infected RBCs (iRBCs) behave similar to leukocytes and undergo margination towards the sidewalls. This provides better understanding of the hemodynamic effects of iRBCs in microcirculation and its contribution to pathophysiological outcome relating to cytoadherence to endothelium. In this work, cell margination is mimicked for the separation of iRBCs from whole blood based on their reduced deformability. The malaria infected sample was tested in a simple long straight channel microfluidic device fabricated in polydimethylsiloxane. In this microchannel, cell margination was directed along the channel width with the iRBCs aligning near each sidewall and then subsequently removed using a 3-outlet system, thus achieving separation. Tests were conducted using ring stage and late trophozoite/schizont stage iRBCs. Device performance was quantified by analyzing the distribution of these iRBCs across the microchannel width at the outlet and also conducting flow cytometry analysis. Results indicate recovery of approximately 75% for early stage iRBCs and >90% for late stage iRBCs at the side outlets. The simple and passive system operation makes this technique ideal for on-site iRBCs enrichment in resource-limited settings, and can be applied to other blood cell diseases, e.g. sickle cell anemia and leukemia, characterized by changes in cell stiffness.

Deformability based cell margination--a simple microfluidic design for malaria-infected erythrocyte separation.

Mass spectrometry (MS) is the enabling technology for proteomics and metabolomics. However, dramatic improvements in both sensitivity and throughput are still required to achieve routine MS-based single cell proteomics and metabolomics. Here, we report the silicon-based monolithic multinozzle emitter array (MEA) and demonstrate its proof-of-principle applications in high-sensitivity and high-throughput nanoelectrospray mass spectrometry. Our MEA consists of 96 identical 10-nozzle emitters in a circular array on a 3 in. silicon chip. The geometry and configuration of the emitters, the dimension and number of the nozzles, and the micropillar arrays embedded in the main channel can be systematically and precisely controlled during the microfabrication process. Combining electrostatic simulation and experimental testing, we demonstrated that sharpened-end geometry at the stem of the individual multinozzle emitter significantly enhanced the electric fields at its protruding nozzle tips, enabling sequential nanoelectrospray for the high-density emitter array. We showed that electrospray current of the multinozzle emitter at a given total flow rate was approximately proportional to the square root of the number of its spraying-nozzles, suggesting the capability of high MS sensitivity for multinozzle emitters. Using a conventional Z-spray mass spectrometer, we demonstrated reproducible MS detection of peptides and proteins for serial MEA emitters, achieving sensitivity and stability comparable to the commercial capillary emitters. Our robust silicon-based MEA chip opens up the possibility of a fully integrated microfluidic system for ultrahigh-sensitivity and ultrahigh-throughput proteomics and metabolomics.

Multinozzle emitter arrays for nanoelectrospray mass spectrometry.

Compared with conventional gel-based techniques, such as gel electrophoresis, which are routinely used for bioseparation in biology and biomedical laboratories, nanofluidic devices with regular engineered sieving structures offer the potential for faster separation, better resolution, higher throughput, and more convenient sample recovery. Here, we detail the fabrication process of a two-dimensional nanofluidic filter array device and its implementation for rapid continuous-flow separation of biomolecules such as proteins.

Nanofluidic devices for rapid continuous-flow bioseparation.

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.

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

High-throughput multiplexed proteomics of small-volume biospecimens will generate new opportunities in theranostics. Achieving parallel top-down and bottom-up mass spectrometry analyses of target proteins using a unified apparatus will improve proteome characterization. We have developed a novel silicon-based microfluidic device, multinozzle emitter array chip (MEA chip), as a new platform for small-volume proteomics using liquid chromatography-nanoelectrospray ionization mass spectrometry (LC-nanoESI-MS). We demonstrate parallel, on-chip, and online LC-MS analysis of hemoglobin and its tryptic digests directly from microliters of blood, achieving a detection limit of less than 5 red blood cells. Our MEA chip will enable clinical proteomics of small-volume samples.

Multinozzle emitter array chips for small-volume proteomics.

The most common markers for monitoring patients with diabetes are glucose and HbA1c, but additional markers such as glycated human serum albumin (HSA) have been identified that could address the glycation gap and bridge the time scales of glycemia between transient and 2-3 months. However, there is currently no technical platform that could measure these markers concurrently in a cost-effective manner. We have developed a new assay that is able to measure glucose, HbA1c, glycated HSA, and glycated apolipoprotein A-I (apoA-I) for monitoring of individual blood glycemia, as well as cysteinylated HSA, S-nitrosylated HbA, and methionine-oxidized apoA-I for gauging oxidative stress and cardiovascular risks, all in 5 μL of blood. The assay utilizes our proprietary multinozzle emitter array chip technology to enable the analysis of small volumes of blood, without complex sample preparation prior to the online and on-chip liquid chromatography-nanoelectrospray ionization mass spectrometry. Importantly, the assay employs top-down proteomics for more accurate quantitation of protein levels and for identification of post-translational modifications. Further, the assay provides multimarker, multitime-scale, and multicompartment monitoring of blood glycemia. Our assay readily segregates healthy controls from Type 2 diabetes patients and may have the potential to enable better long-term monitoring and disease management of diabetes.

Top-down proteomics of a drop of blood for diabetes monitoring.

Biomonitoring of pollutants and their metabolites and derivatives using biofluids provides new opportunities for spatiotemporal assessment of human risks to environmental exposures. Perfluorinated compounds (PFCs) have been used widely in industry and pose significant environmental concerns due to their stability and bioaccumulation in humans and animals. However, current methods for extraction and measurement of PFCs require relatively large volumes (over one hundred microliters) of blood samples, and therefore, are not suitable for frequent blood sampling and longitudinal biomonitoring of PFCs. We have developed a new microassay, enabled by our silicon microfluidic chip platform, for analyzing PFCs in small volumes (less than five microliters) of blood. Our assay integrates on-chip solid-phase extraction (SPE) with online nanoflow liquid chromatography-electrospray ionization-mass spectrometry (nanoLC-ESI-MS) detection. We demonstrated high sample recovery, excellent interday and intraday accuracy and precision, and a limit of detection down to 50 femtogram of PFCs, in one microliter of human plasma. We validated our assay performance using pooled human plasma and NIST SRM 1950 samples. Our microfluidic chip-based assay may enable frequent longitudinal biomonitoring of PFCs and other environmental toxins using a finger prick of blood, thereby providing new insights into their bioaccumulation, bioavailability, and toxicity.

Pan Mao

Newomics, Inc.

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