Een Microfluïdische-gebaseerde Hydrodynamische Trap voor Single Particles

The overall goal of this method is to confine and manipulate single micro and nanoscale particles using laminar flow in a microfluidic device. The hydrodynamic trap consists of a microfluidic device with a cross slot channel geometry, which gives rise to a stagnation point flow at the microchannel junction. An on-chip valve located in one of the outlet channels is used to actively control fluid flow and trap particles.

Particles are confined at a user-defined set point by active control of the stagnation point position. A feedback controller is used to track particle position and to regulate the on-chip valve to maintain particle position at the set point. Using the hydrodynamic trap, single particles are trapped in, dilute or concentrated particle solutions and may be imaged using fluorescence or brightfield microscopy.

A single particle may be confined to within one micron of the trap center as shown in the particle trajectory and the histogram of particle displacement from the trap center. Hi, I'm Eric Johnson Rio from Professor Charles Schroer's Lab in the Department of Chemical and Biomolecular Engineering, and the Center for Biophysics and Computational Biology here at the University of Illinois. Hi, my name is Ari, a postdoctoral researcher at the Schroeder Lab.

Hi, I'm Cheryl Schroeder, and today we're gonna show you how to fabricate and implement a microfluidic device for hydrodynamic trapping of single particles. The main advantage of this technique over existing methods such as optical or electro kinetic traps is that hydrodynamic trapping is achieved by the sole action of fluid flow, thereby eliminating potentially proative four fields for nanoparticle or cell trapping. This technique has the potential to transform fundamental and applied science by enabling free solution trapping of micro and nanoscale particles with no requirements on the chemical or physical properties of the trapped particle.

So let's get started. In order to facilitate peeling the replicas off the SU eight molds, ize the surface of the SU eight molds by placing the wafers in a desiccate under vacuum for 10 minutes with a glass dish containing a few drops of tri chloro seline using mixed and DGAs PDMS for the fluidic and control layers. Spin coat the 15 to one PDMS mixture onto the fluidic layer mold for 30 seconds at 750 RPM, and then place the wafer into a Petri dish.

Similarly, place the control layer mold into a Petri dish and pour a five to one PDMS mixture onto the mold to a thickness of four millimeters. To partially cure the PDMS layers, bake the wafers slash PDMs for 30 minutes at 70 degrees Celsius and allow them to cool to room temperature. Cut the PDMS replica with the scalpel and peel it off the SU eight mold.

Then with the 21 gauge needle, punch a hole in the PDMS as an access port to the microchannel that will act as the on-chip membrane valve. Place the PDMS replica with a control layer onto the wafer with the spin coated PDMS fluidic layer. Carefully align and seal the control layer to the fluidic layer using a stereo microscope.

Make sure to remove all air pockets between the layers and bake at 70 degrees Celsius overnight to fully cure both layers into a monolithic PDMS slab with two layers after cooling to room temperature, use a scalpel to cut and peel the PDMS replica off the SU eight mold and use a razor blade to remove excess PDMS and separate each device unit. Now whole punch access ports to the micro channels in the fluidic layer with a 21 gauge needle. Clean a cover slip with acetone IPA and dry with nitrogen.

Treat both the cover slip and the PDMS replica surfaces with oxygen plasma under 500 millitorr for 30 seconds. And then immediately bring the two surfaces into contact to form an irreversible seal. Finally, bake the devices overnight to increase bonding between the PDMS layers and the cover slip.

First, place the microfluidic device onto the stage of an inverted microscope and secure it with stage clips. Next to deliver the solutions to the microfluidic device, fill a one milliliter and a 250 microliter gast tight syringe with buffer and sample solutions respectively. Use a T valve between the sample syringe and the sample port on the microfluidic device to control sample delivery.

Now establish the fluidic connections between the syringes and the microfluidic device, using per fluoro oxy tubing, lure lock adapters, and 24 gauge metal tubing. Then establish the fluidic connections for the outlet channels in the microfluidic device with PFA tubing and 24 gauge metal tubing. To maintain a constant pressure drop between the syringes and the outlet channels, the PFA tubing for the outlets should be of equal length and both submerged into a 1.5 milliliter centrifuge tube filled with buffer solution.

Fill the on-chip valve with fluorinated carrier oil using a three milliliter lure lock plastic syringe to prevent air from leaking into the fluidic layer during operation. Push the air in the valve chamber through the PDMS membrane into the microchannel. In the fluidic layer.

For on-chip valve operation. Connect a pressurized inert gas supply to the port in the control layer. Rinse the fluidic connections and the microfluidic device with 0.5 milliliters of buffer solution to ensure that all the air bubbles are removed from the system, including the outlet channels.

Typical flow rates used for clearing bubbles range between 2000 to 5, 000 microliters per hour after the air bubbles are rinsed out of the microfluidic channels, reduce the flow rate to 50 to 100 microliters per hour, which is a typical volumetric flow rate for particle trapping. Now switch the T valve to allow sample to flow into the microfluidic device. Execute the custom built lab view code which automates particle trapping by implementing a linear feedback control algorithm.

The code captures images from a CCD camera and transmits an electric potential to a pressure regulator that actively modulates the position of an on-chip pneumatic valve. Using the microscope XY translation stage, position the trapping region at the center of the camera view. Bring the trapping region into the focus of the objective lens and adjust the camera settings to optimize imaging conditions.

Choose a rectangular region of interest within the camera's field of view such that the center of the ROI will be the position of the trap center. Now initialize the offset pressure applied to the on-chip valve. A 100 to 200 micron wide constriction located at the opposite outlet channel.

Provides an offset pressure for the operation of the on-chip valve. Initiate the feedback controller and adjust the proportional gain to optimize trap response. Depending on the flow rate and the on-chip valve position, there is an optimal proportional gain value, which increases trap stability and eliminates unwanted particle oscillations.

The lab view code will automatically trap one of the particles entering the trapping region. In this video particle motion in the inflow direction arises because the sample inlet stream is left open to maximize the trap tightness of confinement in the inflow direction. The user can close the sample stream during trapping, which balances flow evenly into the cross channel junction, monitor the trapped particle and maintain particle focus within the image plane using manual focus or an automated focus microscope setup.

The integrated microfluidic device consists of a sample focus, a cross slot junction and a pneumatic valve trapping occurs at the cross slot junction and the particle position is controlled by active adjustment of the flow field at the microchannel junction through the pneumatic valve. Here, an image of a single bead is confined in the hydrodynamic trap. In addition to the at the trap center, several untrapped beads are shown in the trapping region at the cross lot junction.

The trajectory of a trapped 2.2 micron fluorescent polystyrene bead is mapped. The particle is initially trapped for three minutes. It is then released from the trap and escapes along one of the outlet channels.

A histogram of displacement of a trapped bead from the trap center along the direction of the outlet channels indicates that the particle is confined to within one micron from the trap center. Today, we presented the EM trap as a method for free solution trapping of micro and nanoscale particles using a ation point flow generated within a microfluidic device. Hydrodynamic trapping enables confinement of a single target particle in concentrated particle suspensions, which is difficult to using alternative force field based trapping methods.

After further development, this new technique will allow for scientific exploration in the fields of biophysics, cell mechanics, fluid dynamics, enzymology, and systems biology. Thank you for watching and we hope this technique will be useful for your experiments.