バイオマテリアルのハイスループット検出用高分子マイクロアレイ

This video describes a method for generating and using a polymer microarray using an on-chip photo polymerization technique. First glass slides are dip coated in poly hydroxyethyl, methacrylate, or FEMA to produce a low attachment substrate. Monomers are mixed at varied ratios to produce a library of monomer solutions.

The solutions are transferred to a glass slide format using a robotic printing device and cured with UV irradiation. The monomer polymerizes and the solvent is vacuum extracted. Following high throughput surface characterization, the microarrays can be used for screening for materials performance such as bacterial attachment assays.

Analysis of attachment data is a step towards the discovery of novel biomaterials highlighting the utility of the polymer microarray format. The main advantage of this technique over existing methods where the biological response of different materials are investigated, one material at a time is the parallel screening of hundreds of materials, which are easily manipulated on a single slide. This technique can easily be adapted to various biological assays, such as screening for materials that resist bacterial attachment or support the expansion of stem cells.

This technique has helped discover materials with potential applications for supporting ploy potent stem cell expansion for regenerative medicine. We are also very excited about the recent discovery of materials which resist bacterial attachment with potential to reduce the occurrence of medical device associated infections. Generally, users new to the method will struggle to optimize the conditions required to print a microarray cause it's difficult to predict the optimal conditions.

Getting even small details wrong can lead to non-usable arrays. It is also essential to consider which bacterial strain and the cultural condition will be used because attachment and biofilm morphology are species and the media specific and can therefore vary considerably. Here we use aerogen as a model organism and the chemical defined medium for well controlled attachment assay.

Our MIT collaborators first had the idea for this method through comparison with DNA and protein microarrays. These are well established for exploring their respective biological combinatorial spaces. Dan and Bob realized a similar chemical Combinator space existed that needed to be explored from materials demonstrating a procedure will be Andrew Hook and Chen Chang postdoctoral fellows in our welcome trust funded project Begin this procedure by preparing a low attachment background.

This is a surface that resists the attachment of biomolecules, such as proteins and cells. Weigh out two grams of poly hydroxyethyl methacrylate or FEMA into a 50 milliliter centrifuge tube. Then bring the volume up to 50 milliliters with 95%ethanol in water.

Then place the tube in a ator for about 24 hours or until the FEMA is completely solubilized, holding the glass light with tweezers. Dip in epoxy functional glass. Slide into the FEMA solution to co all but five millimeters of it.

The epoxy groups form covalent linkages with the FEMA coating. The uncoated portion can serve as a positive control over a period of one second. Withdraw the slide from the FEMA solution, then invert the slide and place it in a near horizontal position to dry.

Once the slide is dry, place it in a slide holder. Leave the FEMA coated slides at atmospheric conditions for one week to allow the complete evaporation of the solvent. Next to make the monomer solutions pipette five microliters of photo initiator solution and 15 microliters of dimethylformamide soluble acrylate methacrylate monomers into a 384 well source plate.

A total volume of 20 microliters is ideal for spot formation. Now that the solutions have been generated, a contact printing robot with an X, Y, Z stage is used for generating the microarray. The robot uses 220 micrometer diameter slotted pins that contain slits that function as reservoirs for solution transfer.

Begin by placing the pins and the pin holder in a ator containing di chloro methane for 10 minutes. Then after allowing the pins to dry, reattach the holder to the print head. Next, load the pins into the holder.

Then load 10 glass slides for blotting and FEMA coated slides into the robot. Typically three replicate arrays are printed onto each slide. Then using tubing, connect the robot's wash station to a bottle containing 2.5 liters of fresh dimethylformamide.

Load the plate containing the monomer solution into the robot. Fill the entire chamber of the robot with Argonne to reduce the oxygen level to below 2000 PPM. At this low level polymerization radicals should not be quenched by oxygen.

Using a humidity regulating device, set the humidity to be maintained between 30 to 40%within the robot chamber, including humidity allows for the FEMA to swell, allowing for the formed polymer to interpenetrate the FEMA layer and become physically entrapped to the surface. To start the print run, hit go on the software with the associated computer. The pins will then lower into the monomer solutions at a speed of 25 millimeters per second.

Then they will be held in solution for 2.5 seconds and withdraw at a speed of 25 millimeters per second. Next, the end of the pin is brought into contact with the FEMA slides dispensing approximately 2.4 nanoliters of solution from the quilt part of the pin. The total contact time for each contact is 10 milliseconds and approach and withdrawal speed is 175 millimeters per second.

The pins are then blotted by repeatedly making contact with the glass blotting slides to remove monomer solution from the outside of the pin. The blotting sequence used consists of 33 contacts with a clean glass slide. The first four positions will make 10, 5, 4, and three contacts respectively.

The next four positions will make two contacts, and the last three positions will make one contact Following the printing. The pins are washed in a flow bath of DMF with agitation for 30 seconds. Concurrently the printed slides are irradiated with a shortwave UV 365 nanometer source at a density of 30 millivolts per centimeter squared.

Once the washing and irradiation is complete, the next slide is printed. If the printing looks successful, irradiate the arrays with UV at 365 nanometers for an additional 10 minutes. The freshly printed arrays are placed in a vacuum oven set to less than 50 milli tours for one week to remove un polymerized, monomer and solvent after printing and vacuum extraction.

The success of the polymerization of polymer spots can be assessed by simple light microscopy to identify any anomalous spot morphologies, typically spots should appear circular and uniform as shown in this figure on the left. The likely cause for a change in geometry is a damaged or unclean pin or dust contaminant in the monomer solutions. If this happens, the process will have to be repeated for a small number of monomer combinations.

Misshapen spots may be seen, for example, a central spot with a satellite of small spots shown on the right or a fried egg shape where there is a central spot on top of a large flatter spot. This may be caused by phase separation prior to printing, relating to differences in the viscosity, hydrophilicity volatility or surface tension of the monomers, and suggests that the monomer combination is not compatible with this format. Additional chemical mapping of polymer spots is also an important and sometimes necessary quality control step to determine the distribution of the materials chemistries across the spots and the array high throughput surface characterization or HTSC of the microarrays generated.

Using this technique also allows the biological performance to be correlated with physiochemical properties. This enables the study of biological material interaction. HTSC makes use of water contact angle measurements, time of flight, secondary ion mass spectrometry, atomic force microscopy, and x-ray photo electron spectroscopy.

In particular, TOF sims provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted. The TOF SIM spectra demonstrating the chemical dependence of a biological material interaction and informing the development of HI materials.

Once the polymer microarray surface has been characterized, it can be used for the study of biological interactions. Here we demonstrate the use of the microarray in performing a bacterial attachment assay. Begin by inoculating 10 milliliters of LB with a single colony of GFP labeled pseudomonas.

Aerogen grow at 37 degrees Celsius with 200 RPM shaking for overnight. Measure the OD 600 of overnight bacterial culture and dilute the culture to an OD 600 of 0.01 in 15 milliliters of RPMI 1640. In the meantime, wash the array slides and sterilize distilled water for 10 minutes to remove any dissolvable components from the arrays, such as un polymerized, monomer, all ligaments and solvent.

Then air dry the slides in an airflow cabinet. Next place the washed array slides in a clean Petri dish and UV sterilize them for 10 minutes. When the bacterial culture is ready, transfer the slide with the array into a Petri dish and add 15 milliliters of the inoculated RPMI 1640 media.

Place another array slide in a second Petri dish and add 15 milliliters of sterile RPMI 1640 as a control. Incubate the dishes at 37 degrees Celsius for 72 hours with shaking at 60 RPM following the incubation. Wash the slides three times with 15 milliliters of sterile PBS for five minutes at 100 RPM.

Then wash the slides twice with 15 milliliters of sterile distilled water to remove the phosphate salt. Following the wash. Allow the slides to air dry in an airflow cabinet.

Once the slides are dry. They should be read by a fluorescent scanner as soon as possible to avoid decay of GFP using the appropriate imaging technology and software. The fluorescence due to bacterial attachment is calculated by subtracting the fluorescence from the media only control from the inoculated sample to identify materials that resist bacterial adhesion.

Microarrays were generated and bacterial adhesion assays were performed according to the methods described in this video. As seen here, the polymer microarray contains materials with both low bacterial adhesion, which have low fluorescence and high bacterial adhesion, which have high fluorescence. The three arrays shown here are replicates when the fluorescence values from the three replicates are averaged and presented as a 3D representation.

As shown here, quantitative visual interpretation is simplified. The intensity scale on the right represents the values of the fluorescence data. At a basic level, identifying the materials with low fluorescence provides a means for rapidly identifying materials that fulfill a specific biological application, such as resisting bacterial attachment.

However, these results can also be combined with the HTSC to investigate correlations between particular properties of the materials with their biological performance. This analysis can elucidate structure, function relationships, thus identifying the mechanisms underpinning the biological interaction with materials. After watching this video, you should have a good understanding of how to prepare for and print a polymer microarray and also have an insight into the way the polymer microarrays can be used to probe bacterial response to materials.

Once mastered, a polymer micro array can be printed in two days if performed correctly. The preparation of the monomers takes approximately one day and the printing of the array takes approximately six hours. The bacterial attachment assay takes five days, which increase time to grow the overlay culture as an inocular In incubating the microray light in the bacterial culture for 72 hours and detecting the forest and signal using microray scanner, Okay, while attempting this procedure, it's important to keep the pin and the pin holders clean.

It's very frustrating if halfway through a run the pin gets stuck in an upright position and can no longer make contact with the surface cause the pin or the pin holder wasn't sufficiently cleaned. Don't forget that working with DMF and toxic monomers can be extremely hazardous and precautions such as the extraction should always be included in the procedure.