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 scientific approach signaling pathways modeling microsystems microsystems in-depth soft lithography measurement scientists  |
Microsystems In-DepthMicrosystems in-depth
MicrosystemsKlavs Jensen IntroductionExisting methods for sample preparation and detection are disparate. Thus, the amount of data used for predictive models of signaling networks is currently limited by the labor associated with the measurement process. Our ultimate goal is to create a microfluidic system that converts a continuous input of cells into a real-time and quantitative output of protein concentration and modification state, and thereby directly monitor the dynamics of biomolecular networks as they unfold. We have thus far validated the individual components that will ultimately be integrated together to create the microfluidic system. These components exploit unique physical principles associated with micro- and nanofluidic channels to achieve fidelity that will ultimately match or exceed the performance of existing methods. AccomplishmentsCell stimuli and lysis Signaling events can exhibit fast transient responses (15-45 sec) and successful analysis requires very short mixing times with well controlled and reproducible stimulus conditions. Such time resolution can be difficult to reproducibly measure with conventional methods since small fluctuations in the manual handling become significant at short times. While microfluidic devices offer the potential for reproducible and automated analysis, the laminar flow conditions make it difficult to achieve fast mixing times. We have addressed this challenge by developing a novel microfluidic device that uses enhanced convective mixing in segmented gas-liquid flow to achieve sufficiently short mixing times to resolve fast transient responses in cell signaling pathways. Stimulation of cells performed in the device resulted in comparable pathway activation to that using conventional methods, and the good time control in the device resulted in reproducible analysis of fast transient responses in the cell signaling pathways. Separation We have demonstrated a new approach for size-based separation in microfluidics that does not require conventional sieving matrices. The approach is based on Ogston sieving mechanism where nanofilter arrays with gap sizes of 40-180 nm are used to separate proteins and DNA molecules according to their size. When compared to sieving matrices, nanochannels are i) easier to integrate into microfluidic systems containing sensors and reaction chambers, and ii) chemically and mechanically robust and can be used over a long period of time without degradation. We have separated SDS-protein complexes within several minutes and have shown that the separation efficiency is comparable to state-of-the-art systems based on capillary gel electrophoresis. We have also developed a qualitative and quantitative understanding of Ogston sieving that will provide design guidelines for further improving separation efficiency for a wide range of biomolecules. We are also developing microfluidic devices that use free flow isoelectric focusing (IEF) to separate proteins and sub-cellular organelles based on their isoelectric point. However, the fidelity of the separation is often limited by the ability to apply sufficiently high electric fields across the width of the microchannel. We have taken a first step towards addressing this limitation by creating an electrical interface with nanoporous and hydrogel materials (“soft electrodes”). These electrodes allow for electric potentials that are two orders of magnitude greater than what is possible with patterned metal electrodes. Concentration We have developed a novel nanofluidic device that can achieve more than a million-fold sample preconcentration within an hour. The entire system consists of two microfluidic channels (a few tens of µm in dimension) bridged by a nanofluidic channel as thin as 40nm in depth. At moderate buffer concentrations (~10mM), the Debye layer thickness within a nanofluidic channel is not negligible, and the nanofluidic channel becomes perm-selective when an electric field is applied across the nanochannel. The mechanism of the nanofluidic concentrator can be explained by nonlinear electrokinetic phenomena. To date, we have demonstrated preconcentration of peptides, proteins, and DNA molecules. There are many characteristics that make this device ideal as a component for integrated sample preparation: i) the concentration factors achieved in this device are exceptionally high, probably due to the fact that one can concentrate the dilute sample for a long time. The stability of the system is partly due to the mechanical robustness of the solid-state nanofluidic filter membrane. ii) The operation of the device is not dependent on the specific kind of buffer solution or any reagents used. iii) The concentration occurs in a microfluidic channel that is comparable to the size of a capillary system, without using any membranes or filters blocking the flow of concentrated solution. Therefore, the nanofluidic channel does not limit the flow rate or the capacity of the device since it is simply providing an energy barrier. Detection We have demonstrated a fundamentally new approach for detecting biomolecules in the aqueous environment. Known as the suspended microchannel resonator (SMR), target molecules flow through a 10 pL suspended microchannel and are captured by receptor molecules attached to the interior channel walls. As with other resonant mass sensors, the SMR detects the amount of captured target molecules via the change in resonance frequency of the channel during the adsorption. However what separates the SMR from the myriad of existing resonant mass sensors is that the receptors, targets, and their aqueous environment are confined inside the resonator, while the resonator itself can oscillate at high quality factor Q in an external vacuum environment, thus yielding extraordinarily high mass resolution. Initially, SMR devices were fabricated at MIT facilities and packaged at the level of individual devices with PDMS microfluidics. While this approach led to a successful demonstration, the PDMS packaging process was tedious and the overall system was delicate, unstable, and difficult to reproduce. To address these limitations, we established a partnership with Innovative Micro Technology (IMT) to implement a packaging process based on full-wafer, bonded glass microfluidics. The SMR devices are fabricated at MIT and then sent to IMT for packaging and dicing. The resulting devices yielded a sensitivity that approached 10-18 g/um2 which is nearly an order of magnitude more sensitive than the commercial quartz crystal microbalance. Future plansOur efforts are being directed towards two areas: i) optimization and application of an individual component for assays relevant to the CDP, and ii) integration of multiple components to demonstrate a level of performance that cannot be achieved by individual components alone. In the first area, we are developing protocols that enable the specific detection of unlabelled Akt by using the suspended microchannel resonator. Antibodies will be covalently attached to the detector walls and mass increase upon binding of Akt to the immobilized receptors will be measured directly by the change in resonant frequency. We are also developing low noise electronics readout for the microchannels that are scalable, suitable for mass production, and extremely robust. Such circuitry will enable microchannel arrays to detect the abundance and state of multiple signaling proteins in parallel. In the second area, we are integrating our microfluidic segmented flow device with protein microarrays to measure Akt abundance with a fluorescent sandwich assay. In a separate project, we are integrating the electrokinetic concentrator with an affinity assay for Akt. For the short-term, we will use the fluorescent sandwich assay, and in the long-term, we will integrate the concentrator directly with the suspended microchannel resonator for label-free detection. We anticipate that such a device will provide a general approach for improving the sensitivity and dynamic range of immunoassays. Since the gain of the concentrator is adjustable, the dynamic range and detection limit of the immunoassay will ultimately be governed by the properties of the concentrator and not the binding affinity constant. References
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