マイクロ流体液滴のプラットフォームのための蛍光検出法

Hello, I'm Jersey. Hi, my name is Shabi and we are both postdoctoral researchers at Imperial College of London, working with Professor Andrew Di Melo and Dr.Josh Del in microfluidic droplet platforms. Droplet based microfluidic platforms promising candidates for higher throughput experimentation because they can be generated at a very high precisely defined volumes and their competitions, the generation frequency can be as high as several kilo pers.

Furthermore, since the region of interest can be encapsulated in compartments as later as a few liter, both crosstalk and dispersion can be eliminated, and analytical processes can be timed with greater accuracy. In our group, we have already successfully implemented micro droplet platforms to a lot of applications. For example, polymerase chain reaction, nanoparticle synthesis, and the single molecular cell assay.

The large amounts of information generated within these systems must be efficiently extracted, harnessed, and utilized. In this respect, the detection method of choice should be right, provide high sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive, and be able to be integrated with microfluidic devices in a facile manner. To address this need, we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of for physical information from small volume environments, and are applicable to the analysis of a wide range of physical, chemical, and biological tors.

In this protocol, we will be presenting to methods which are applied to the detection of single cells and the mapping of mixing processes in Dropbox. Microfluidic chips are traditionally fabricated in poly dimethyl Sloane PGMS using photolithography methods. This process ought to be carried out in a clean room facility to obtain microfluidic channels Spinco, SU eight 50 on a silicon wafer soft bake on a hot plate at 65 degrees and 95 degrees to evaporate the solvent and to densify the film after cooling down, expose the SU eight, resist with UV radiation under the appropriate mask, and then bake the wafer at 65 degrees and 95 degrees after cooling.

Develop the unexposed areas using the appropriate developing solution while stirring. Use fresh EC solvent to rinse the wafer and then dry it under gas to generate a clean surface. Treat the wafer with hexane and methanol washes.

Also to complete the esate master fabrication process, evaporate a seizing agent onto the surface in a desiccate for 40 minutes. To form structured microfluidic substrates, we mix S guard 1 8 4 in a 10 to one ratio of resin crosslinker and pull this over the master. Degas this in a desiccate and cure the device at 65 degrees.

Cut the cured PDMS and peel it off the master. Create access holes for fluidic tubing using a biopsy punch. Cure another layer of PDMS for 20 minutes at 65 degrees.

Place the prepared top layer on the bottom layer and cure overnight at 65 degrees. Peel the chips from the Petri dish and dice. Once the chips have been fabricated, they're ready to be used by infusing the solutions of interest.

Fill syringes with the required solutions, ensuring no air bubbles remain in any parts of the tubing. For droplet generation, two phases are used. The aqueous dispersed phase contains the reagents of interest.

The oil phase acts as the continuous phase fit tubing of the same internal diameter as the syringe needle tips on one end to the needles mount the syringes on a syringe pump and define the desired infusing flow rate. For each phase, a minimum ratio of oil aqueous flow rates of one is recommended to avoid wetting of the PDMS by the aqueous phase, which would lead to unstable droplet formation. Connect the other ends of the tubing directly to the holes punched in the PDMS substrate.

For more complex solutions, silica, capillary ports or connectors can also be implemented. Connect the outlet port of the chip to the tubing and direct into a collector for waste collection or further analysis processing. Once this is set up, there are two main configurations of microchannels to generate droplets flow focusing or a T junction format as a result of the sheer forces that arise from using these geometries to bring the two admissible fluids together and the subsequent capillary ins that develop in the interface.

One oh disperse droplets as small as a few femtoliters are generated spontaneously. Once the droplets are being generated, they can be probed using an optical setup. The optical system is set up the following protocol.

Use a confocal microscope with automated submicron positioning capacity and a laser operating at a wavelength that matches the absorption of the Fluor falls involved. To excite them, use a pulse laser operating at repetition rates of several megahertz for fluorescent lifetime imaging. Phlegm experiments use beam steering mirrors and optics to control the beam height as well as beam direction.

Use a dichroic mirror to reflect the laser beam into the objective lens. If two lasers are used simultaneously, a dual band diic mirror can be installed. To allow reflection of the two laser beams, use an infinity corrected high numerical aperture NA microscope objective to bring the laser light to a tight focus within a microfluidic channel.

Collect fluorescence emitted by the sample within the microfluidic channel, using the same high NA objective and transmitted through the same dichroic mirror. Remove any residual excitation light using a single or dual band emission filter. Focus the fluorescence emission onto a 75 micrometer pinhole.

Using a plano convex lens. Position the pinhole in the confocal plane of the microscope objective. Use another dichroic mirror to split the fluorescent signal onto two detectors.

Filter the fluorescence reflected by the dichroic mirror with an emission filter and focus it with a plaino convex lens onto the first detector. For experiments involving a secondary red Fluor, filter the fluorescence transmitted by the diic mirror with another emission filter, and then focus it with another plano convex lens onto the second detector. Use avalanche photo diodes for detection of fluorescence photons.

For the fluorescence data acquisition, couple the electronic signal from the avalanche photo diode detector to a multifunction DAQ device for data logging running on a personal computer. For phlegm experiments, use a pulse laser and connect the detectors to a time correlated single photon counting TCS PC card running on a separate pc. This card allows for the fluorescent lifetime data to be obtained.

Using a TCS PC methodology. Each detected fluorescent photon is correlated to an incident laser pulse and therefore each emitted fluorescent photon is assigned a delay time. This data can be fitted to a single or multi exponential decay curve to obtain an estimation of the fluorophores radiative rate.

We now know that cells are highly heterogeneous from both biological, physical and a chemical perspective. Therefore, ideally, we should analyze each cell at a time. However, the assays should be performed with a higher throughput so that we can collect enough quantitative information to come to a reliable and meaningful conclusion.

Here we have an example of a encapsulating e coli cells in single droplet, so that as is can be performed. Use e coli top 10 strain that has been cultured overnight. In order to detect the viability of the cells, use cyto nine and propidium iodide.

Both of the dyes are DNA inter collating dyes and their fluorescence intensity increases by over 20 folds. Upon binding to DNA cyto nine is a green fluorescent dye that is membrane permeable and propidium iodide. It's a red fluorescent dye, which is membrane impermeable.

Thus live cells fluoresce green while dead cells exhibit both green and red emissions. Here we can see droplet generation and a single cell encapsulation with a T junction. This video was recorded with a high speed camera, but droplets can be generated at a rate of thousands per second.

When encapsulating cells, one must make sure that the culture of the cells is diluted so that there are fewer cells than droplets generated, that the cells are prevented from sedimenting, which can occur in the syringe or tubing. Use a magnetic stir bar inside the syringe to keep the cells evenly distributed in the solution. Use very thin tubing, 50 micrometers to connect the syringe to the microchip.

This ensures a fast flow rate inside the tubing and therefore prevents the cells from sedimenting. Here are the typical examples of photo counts as a function of time for the cell-based experiments. To map mixing processes within droplets.

Droplets can be generated using two different fluoro fours with different lifetimes. Within a flowing droplet, a flow field is generated, which eventually mixes the two originally separated solutions to enhance the mixing process. The symmetry of the flow field can be altered by using winding channels.

In order to probe the droplets, focus the optical probe volume at half the height of a microchannel along which droplets are flowing, beginning from one side of the channel, carry out each experiment along the entire width of the channel. At one micrometer intervals. Implement an algorithm to differentiate single bursts associated with droplets from the noise background of the oil phase, and to establish the duration of each burst.

Implement a second algorithm to extract the delay time and intensity values along the length of each droplet at the particular width where the experiment has been carried out. Then use a maximum likelihood estimator MLE algorithm to evaluate the fluorescence lifetime of each droplet in the experiment. Averaging the lifetime values for all the droplets in the experiment reduces the final error of the MLE calculation.

The more droplets probed, the smaller the error once a lifetime trajectory has been obtained. For each width, combine all the trajectories in a 2D map. Since each lifetime value is associated with a particular mixture of the two flora fours, a concentration or mixing map can thus be obtained.

We have described the fabrication of a micro device and experimental set up and associated protocols for micro droplet formation and the region encapsulation and the optical detection of the process, the droplets. The two examples selected the detection for single cells in droplets and the mapping of the mixing processes. Inside the flowing droplet represent common applications that are currently being investigated with the droplet microfluidics.

As the presented experiments illustrate the use of droplet microfluidic platforms present certain attractive characteristics such as high throughput cause the perfect confinement and higher reproducibility. The large amount of information produced and the highest speed at which this information is being generated. DU required the use of fast detection methods with highest spatial temporal resolution if the full advantage is to be gained from this miniaturized platforms.

In this case, we demonstrate that this is possible using highly precise fluorous spectroscopic techniques.