A Reliable, Label Free Quality Control Method for the Production of DNA Microarrays with Clinical Applications

Abstract

The manufacture of a very high-quality microarray support is essential for the adoption of this assay format in clinical routine. In fact, poorly surface-bound probes can affect the diagnostic sensitivity or, in worst cases, lead to false negative results. Here we report on a reliable and easy quality control method for the evaluation of spotted probe properties in a microarray test, based on the Interferometric Reflectance Imaging Sensor (IRIS) system, a high-resolution label free technique able to evaluate the variation of the mass bound to a surface. In particular, we demonstrated that the IRIS analysis of microarray chips immediately after probe immobilization can detect the absence of probes, which recognizably causes a lack of signal when performing a test, with clinical relevance, using fluorescence detection. Moreover, the use of the IRIS technique allowed also to determine the optimal concentration of the probe, that has to be immobilized on the surface, to maximize the target recognition, thus the signal, but to avoid crowding effects. Finally, through this preliminary quality inspection it is possible to highlight differences in the immobilization chemistries. In particular, we have compared NHS ester versus click chemistry reactions using two different surface coatings, demonstrating that, in the diagnostic case used as an example (colorectal cancer) a higher probe density does not reflect a higher binding signal, probably because of a crowding effect.

Keywords: DNA microarray; label-free detection; spotting optimization.

Figure 1

Structure of MCP-4 and Copoly Azide 10% copolymers. Both copolymers have a backbone of N,N-dimetheylacrilamide, but while MPC-4 contains N-acryloyloxysuccinimide as functional groups, which allows nucleophile substitution with amines, Copoly Azide 10% bears azide moieties for click chemistry reactions with alkyne or DBCO groups.

Figure 2

Scheme of the assay. (a) In the case of KRAS wild-type allele, the Stabilizer oligonucleotide (green rectangle) hybridizes in solution with the synthetic single strand PCR (ssPCR) to keep the synthetic ssPCR sequence stretched, then the Dual-Probe 5’ domain (yellow rectangle), complementary to the KRAS wild-type synthetic ssPCR, hybridizes in solution with its specific sequence inside the synthetic ssPCR. (b) The sequence in the 3’ domain of the Dual-Probe oligonucleotide (red rectangle) directs the synthetic ssPCR to its complementary capture probe on the array (dark red rectangle). The position in the array is revealed when the U-tag sequence at 5’-end of the synthetic ssPCR (blue rectangle) interacts with the complementary Cy3-labeled oligonucleotide, Universal-Cy3 (U-Cy3), added in the last step of the assay.

Figure 3

Scheme and images of the multiplexed chip utilized for IRIS quality control assay. (a) Spotting scheme of the 33 oncogene capture probes, where different colors indicate a different codon or gene. (b) IRIS image of a chip where KRAS-12 C (red rectangle) and NRAS-61-ht (green rectangle) were absent due to poor immobilization. (c) IRIS image of a new chip where immobilization for KRAS-12-C was optimized (red rectangle).

Figure 4

Binding curves obtained with the Interferometric Reflectance Imaging Sensor (IRIS) system for the specific capture of DNA target sequences. The five samples at 50 nM (NRAS WT, KRAS WT, G12C, G12D, G13D) were injected sequentially into the fluidic chamber and bound stably to their complementary probe (NRAS WT, KRAS WT, KRAS 12C, KRAS 12D, KRAS 13D, respectively, thick lines). No non-specific binding was detected on any of the other probes (thin lines).

Figure 5

Spotting scheme on (a) MCP-4 and (c) Copoly Azide 10% coated surfaces and IRIS images of the (b) MCP-4 and (d) Copoly azide 10% chips, taken immediately after spotting.

Figure 6

Immobilized mass of the probes spotted on (a) MCP-4 and (b) Copoly Azide 10% coated chips, obtained by analyzing the IRIS images of Figure 5.

Figure 7

Fluorescence signal intensities obtained after hybridization of the spotted probe with a synthetic ssPCR. Fluorescence detection has been obtained using a Cy3-labelled oligonucleotide (U-Cy3) complementary to the U-tag sequence at 5’-end of the synthetic ssPCR. The same experiment was performed on (a) MCP-4 coated chips, onto which amine modified oligonucleotide were immobilized, and on (b) Copoly Azide 10% Coated chips; in this case DBCO modified probes were used. Fluorescence was detected using a confocal laser scanner, setting the power to low and the gain to 1%.

Figure 8

Binding curves obtained with the IRIS system for complementary targets binding to the respective probes at different concentration on a (a) MCP-4 (b) Copoly Azide 10% coated surface.