Fabrication of DNA polymer brush arrays by destructive micropatterning and rolling-circle amplification

Abstract

A method for fabricating DNA polymer brush arrays using photolithography and plasma etching followed by solid-phase enzymatic DNA amplification is reported. After attaching oligonucleotide primers to the surface of a glass coverslip, a thin layer of photoresist is spin-coated on the glass and patterned via photolithography to generate an array of posts in the resist. An oxygen-based plasma is then used to destroy the exposed oligonucleotide primers. The glass coverslip with the primer array is assembled into a microfluidic chip and DNA polymer brushes are synthesized on the oligonucleotide array by rolling-circle DNA amplification. We have demonstrated that the linear polymers can be rapidly synthesized in situ with a high degree of control over their density and length.

Figure 1

Fabrication of DNA polymer brush arrays. Linear DNA polymer arrays are fabricated on glass coverslips using a destructive micropatterning technique and RCA.

Figure 2

Microfluidic device with temperature control. (A) Exploded view of the device with temperature control and integrated fluidics for automated heating, cooling, reagent loading, and washing; a: polycarbonate block with fluidic ports; b: glass slide with drilled holes; c: double-coated silicone adhesive tape; d: glass coverslip containing the DNA primer array; e: aluminum heating and cooling plate; f: thermoelectric modules; g: aluminum heat sink with channels for water cooling. Leak-free connections are made between the glass slide and the polycarbonate block using silicone O-rings (not shown). (B) Photograph of the assembled device.

Figure 3

High-density arrays of DNA polymer brushes. (A) Fluorescence image of a portion of an array of linear DNA polymers synthesized using RCA on an array of DNA primers; (B) Close-up view of a portion of the polymer array containing a missing polymer cluster. These dropouts occur when the posts in the photoresist are lost during development. The subsequent plasma treatment results in primer destruction in these unprotected regions; (C) Surface plot of the polymer clusters shown in (C); (D) Close-up view of a portion of the polymer array after a 30 min RCA reaction. With extended synthesis times, the linear polymers can become long enough to bridge adjacent clusters.; (E) Surface plot of the polymer clusters shown in (D). The scale bar is 40 μm in (A) and 3 μm in (B) and (D).

Figure 4

Control of the density of DNA polymer brushes. Fluorescence images showing the effect of circular DNA template concentration on the density of the DNA polymer brushes. The circle concentrations were varied from 200 to 0.2 × 10⁻⁹ m: (A) 200 × 10⁻⁹ m, (B) 20 × 10⁻⁹ m, (C) 2 × 10⁻⁹ m, and (D) 0.2 × 10⁻⁹ m. The individual polymers can be resolved at the lower concentrations (C and D). These fluorescent micrographs were acquired with a 40× NA = 1.3 oil objective and an EMCCD camera with 8 × 8 μm² pixels. The scale bar is 15 μm.

Figure 5

Analysis of DNA polymer synthesis by enzymatic digestion and gel electrophoresis. (A) An image of a 20% polyacrylamide gel showing the result of an enzymatic digestion of the linear DNA polymers synthesized via solid-phase RCA. The primary bands in the right three lanes are 78-base fragments corresponding to the length of the circle used to synthesize the polymers. The higher bands correspond to fragments that are multiples of the 78-base fragment and are a result of incomplete digestion. The lower bands, which are 14 and 23 bases in length, are the digested products of the 37-base oligonucleotides that were hybridized to the polymers to introduce the restriction enzyme cutting sites. (B) A plot of the total amount of DNA synthesized per flow cell as a function of RCA reaction time. The growth rate is approximately linear in this range (Y = 7.6X + 19.2, R² = 0.99).

Figure 6

Analysis of the DNA polymer length by enzymatic digestion and gel electrophoresis. (A) An image of a 0.5% agarose gel showing the average linear DNA polymer lengths for three different time points. The polymers were synthesized via solid-phase RCA, converted into a double-stranded form and then released from the substrate by cutting the molecules near the base with a restriction enzyme. (B) A plot of the average polymer length in kilobase pairs as a function of RCA reaction time. The polymer growth rate is approximately linear in this range (Y = 2.1X + 2.9, R² = 0.98).

Figure 7

Characterization of brush polymer arrays by AFM. (A) An AFM image of a 12 × 12 μm² portion of the polymer array. The array was produced by performing a 3-min RCA reaction after the hybridization of the 78-base circle at a concentration of 2 × 10⁻⁸ m to the primers on the surface. (B) Height profile of one feature of the brush array. The profile corresponds to the line shown in (A).