Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches

Abstract

A novel method is proposed for direct detection of DNA hybridization on microarrays. Optical interferometry is used for label-free sensing of biomolecular accumulation on glass surfaces, enabling dynamic detection of interactions. Capabilities of the presented method are demonstrated by high-throughput sensing of solid-phase hybridization of oligonucleotides. Hybridization of surface immobilized probes with 20 base pair-long target oligonucleotides was detected by comparing the label-free microarray images taken before and after hybridization. Through dynamic data acquisition during denaturation by washing the sample with low ionic concentration buffer, melting of duplexes with a single-nucleotide mismatch was distinguished from perfectly matching duplexes with high confidence interval (>97%). The presented technique is simple, robust, and accurate, and eliminates the need of using labels or secondary reagents to monitor the oligonucleotide hybridization.

Figure 1

End-point DNA hybridization detection. (a) The original image of the array is seen after washing the spotted substrate. The 360 spot array contains 4 replicate arrays; in each array, every DNA sample was spotted as 10 replicates. (b) The difference image after hybridization clearly shows mass increase on the specific spots and no hybridization on the control spots of 40(−) and 20ds. (c) Average mass densities on the spots measured before and after hybridization for different DNA strands are shown (n=40). Error bars represent +/− 1 standard deviation of measured mass densities among the spots. (d) Percent mass change as a result of hybridization. Error bars represent +/− 1 standard deviation of detected mass change among the replicate spots. For the perfectly matched duplexes, hybridization is clearly detected and the efficiency is close to 100%.

Figure 2

The dependence of DNA denaturation kinetics on ionic buffer concentration. (a) 30 spots per probe type are averaged in the denaturation curves. The denaturation of 18DM strands starts when the salt concentration of the buffer is about 2.5 mM, indicating that it is the least stable duplex that is being tested. Incubation with a buffer of 0.625 mM salt concentration initiates denaturation of all duplexes. Notice that the total drop in mass density depends on how much initial hybridization was present, thus, the mismatches cannot be distinguished by the level of mass density drop. However, how fast this drop occurs gives information about duplex stability. (b) The images from the real-time experiment show denaturation patterns of the different duplexes. Since the data at t=80 min. is subtracted as a reference, the spots are only visible if there is mass change. The spots begin to darken indicating a decrease in the mass density as denaturation occurs. The images correspond to time points (from left to right): t=100 min.; t=110 min.; t=120 min.; t=160 min.; t=190 min.

Figure 3

Kinetic characterization of DNA denaturation. Data for 15 individual spots of 20PM, 20MM and 20DM are shown for demonstration; kinetic data belonging to other samples were processed identically. Single decaying exponentials were fit to each of the denaturation curves for the time period between t₁=105 min. and t₂=185 min., to find the decay constants. In the inset, example fits of randomly selected 4 spots from 20PM samples and 4 spots from 20MM samples are shown by plotting both their kinetic data and the corresponding fits (shown in red).

Figure 4

Denaturation decay constants of DNA duplexes. (a) Decay constants of 20PM and 20MM are compared; (b) decay constants of 18PM and 18MM are compared. A higher decay constant indicates a faster denaturation, thus a less stable DNA duplex. In both cases the single mismatch is clearly detectable. Having a better separation on the 18 nucleotide long sequence indicates a higher impact of the mismatch on the sequence stability. (c) Decay constants of 20PM and 18PM are compared. Even though both of the duplexes were formed by perfectly matching sequences, the shorter strands form slightly less stable duplexes. (d) The similarity of the 20ds and 20PM decay kinetics indicates that the strands that were hybridized prior to spotting and the strands that were hybridized on the chip have the same stability, as expected.