Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

DNA biochips are an attractive research platform for self re-synthetic biology, originally developed by the BASF group, which allows for the operation of in vitro gene circuits over extended periods of time. With this protocol, we hope to make this technology available to a wider range of researchers. Our method, termed bephore lithography, represents a strategy for the immobilization of DNA based on the DNA strand displacement reaction.

The strength of this technique lie in its multi-step lithographic capabilities, its simple reproducibility, and the commercial availability of all the components. Similar to other biochip solutions, bephore can be combined with different technologies. For example, functional gene brushes can be assembled within micro-compartments for protein expression.

To begin, prepare an RCA mixture by mixing five parts water with one part of a 30%ammonia solution in a glass beaker. Heat the mixture to 70 degrees Celsius on a hot plate while stirring. Once it reaches 70 degrees Celsius, add one part 30%hydrogen peroxide.

Then place a 100 millimeter diameter silicon wafer into the solution, to remove organic material and particles from it. After 30 minutes, take out the substrate, rinse it thoroughly with water from a wash bottle, and dry it with a nitrogen gun. Immediately proceed with the PEGylation of the substrate.

To accomplish this, first dilute biotin-PEG-Silane in dry toluene to 5 milligrams per milliliter, and vortex the solution until it is well mixed. With the substrate placed in a glass Petri dish, slowly pipette the solution onto the substrate, covering the whole surface, but avoid allowing the solution to flow over the edge. Once the surface is covered, close the Petri dish, and then add an additional cover.

After 30 minutes, remove the cover, and add approximately 40 milliliters of isopropyl alcohol to the Petri dish. Then take out the substrate, rinse it again thoroughly with isopropyl alcohol, and dry it with a nitrogen gun. When dry, store the substrate in the dark until it is needed.

Prepare a substrate by using a glass cutter or a scalpel to form one centimeter by one centimeter pieces. Then, add a simple alignment mark by making a short scratch from the center towards an edge of the substrate. Blow any small particles off the chip using a nitrogen gun.

Next, mix two droplets of a two-component silicone glue, and apply the glue to the surface of the chip, leaving the area around the tip of the scratch blank. There the glue provides a hydrophobic barrier, keeping aqueous solutions on the chip. This facilitates subsequent washing steps, and reduces the amount of DNA required for incubations.

In a later step, the glue can be easily peeled off. In the following steps, photocleavable DNA is handled only in a yellow light environment. Cover the chip with about 10 microliters of a bephore mixture at room temperature, and place the chip in a box partially filled with water to reduce evaporation.

After one hour, wash the chip several times with PBS to remove any unbound bephore mix. Next, add 10 microliters of the passivation mix to the chip. After two hours, wash the chip several times with PBS to remove the unbound passivation agents.

Cut a printed photo mask to the appropriate size in order to fit a suitable mask holder. Insert it at the position of the field stop, and use the alignment marks to align the mask in the holder. With the substrate placed on the microscopy stage, insert a red filter into the illumination path, and focus onto the substrate surface.

Then, navigate to the region of the substrate which will be exposed. Next, insert the mask holder, block the illumination, and change to the UV filter. At a low light intensity, open the shutter, and use the camera to quickly focus the mask on the substrate.

With the substrate now aligned and the mask in focus, block the light path to the camera, and illuminate the substrate at a high light intensity for the desired exposure time. After exposure, remove the sample from the microscope, and carefully remove as much buffer as possible from the substrate without drying it. Then, add 10 to 20 microliters of DNA with the sequence for attachment to the surface.

Place the substrate in a wet box to keep it from drying, and incubate the DNA on the substrate for two hours at room temperature. In order to observe the compartmentalized gene expression on an inverted microscope, first separately prepare the two parts of the sample holder. Start by gluing a bephore chip with an immobilized gene brush onto the chip holder, using double-sided adhesive tape.

Next, add some vacuum grease around the central hole of the bottom part of the holder, and insert the chip holder. Now assemble the top part of the holder. Cut a thin PDMS chip with compartments as small as possible, leaving a channel open to one side to later allow for the exchange of waste and precursor molecules by diffusion Next, plasma-treat a glass cover slip in the back side of the PDMS chip with oxygen plasma.

Immediately after treatment, place the PDMS chip at the center of the glass slide, with the compartments pointing upwards. Then, bake the glass with the PDMS for one hour at 70 degrees Celsius. Shortly before assembling the entire sample holder, plasma-treat the glass slide with the PDMS chip as before.

Then, add some vacuum grease around the large hole of the top holder, and place the glass slide with the PDMS onto it. Gently press the glass onto the grease. Carefully remove the buffer from the chip, and wash it once with 10 microliters of self re-expression system.

Next, add 60 microliters of self re-expression system onto the chip, and remove the two-component silicone glue from the edges. Now, add 20 microliters of the expression system onto the PDMS. After placing the droplet onto the PDMS, quickly check in the stereoscopic microscope that the compartments are well wet and without air bubbles.

If there are air bubbles, try to wash them away. To assemble the two pieces of the holder, and to align chambers and DNA brushes, work under a stereoscopic microscope. Immobilize the bottom holder with a gripper arm so the two screws and wingnuts will be easily accessible with both hands.

Insert the top holder into the bottom holder, and lower it until the cell-free expression system droplets fuse. Then, check to make sure the compartments and the alignment mark are in a similar region in the XY plane. From the bottom, screw up the wingnuts until they touch the bottom side of the holder.

While aligning the compartments and the chip in the XY plane, gently tighten the wingnuts. This step requires some experience, and depends on the construction of the sample holder. The PDMS should be pressed onto the chip only with gentle force.

Next, spray a sponge in the anti-evaporation enclosure with water, and insert the holder into the box. Then, fill a five milliliter syringe with two-component silicone glue, and use it to seal the box. Finally, transfer the box to a temperature-controlled microscope, and image the DNA brush and the reaction using fluorescence microscopy.

A two-step lithographic process, here performed on a bephore glass slide, yields overlapping patterns of fluorescently labeled DNA strands. In this demonstration of on-chip gene expression, DNA is immobilized in the chamber on the left. When exposed to the expression mix, the yellow fluorescent protein YPet is synthesized from the immobilized DNA.

To assess the activity of the gene expression system, the recovery of the fluorescence is observed after a bleaching step. At two hours, the fluorescence intensity recovered quickly. However, after four and six hours, it did not recover, indicating that without a fresh supply of the expression mix, the reaction terminated around hour four.

Although gene expression is limited in a closed system, it can be sustained over longer periods of time by supplying the expression compartments with additional precursor molecules via micro-fluidics. The bephore method is used to immobilize oligonucleotides or gene length DNA, which can be applied to construct systems of interacting DNA brushes for self re-gene expression. The technique may also be used for other applications in biophysics, such as single molecule fluorescence studies.

After watching this video, you should be able to fabricate bephore biochips from wafers or glass slides, and integrate them into your research projects.