Label-free Detection of Oligonucleotide Microarrays by the Scanning Kelvin Nanoprobe

The Kelvin measurement is a sensitive and label-free method based on work function measurements. Work function, the minimum energy required to extract an electron from a metallic material, can be shifted by ionic charges and dipoles present on the surface. The scanning Kelvin nanoprobe (SKN), a probe-based microscopic imaging device, was used in the detection of work function changes induced by surface-immobilized oligonucleotide / DNA microarrays. The scanning Kelvin nanoprobe was able to study DNA microarrays smaller than 100 µm in size, produced with solution concentrations lower than 10 µmol/L. The limit of detection was estimated to be 15 ng DNA. Better than ± 10% relative variation was achieved for replicate spots. It was observed that higher surface densities of immobilized DNA molecules produced greater work function changes than lower surface densities. Surface saturation with increasing solution concentrations was observed as well. Also, longer strands of DNA produced greater work function changes than shorter strands. Statistical analysis of the results confirmed that non-complementary DNA strands could be differentiated from complementary strands by the Kelvin measurement. Single base mismatches on the complementary DNA strands were also detected by the Kelvin measurement. Different substrate materials were tested in the search for reliable and inexpensive sample slides with satisfactory DNA immobilization efficiency. Materials such as silicon wafers, gold-coated glass slides, gold-coated stainless steel slides, and gold compact discs (CD) were tested. A surface property comparison of gold-coated glass slides and compact discs was made by atomic force microscopy (AFM), and revealed very different microscopic features. The effect of cleaning on gold-coated glass slides was examined by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Technical improvements were made to the SKN equipment progressively. Several revisions to the tip holder design have been employed for better electromagnetic shielding, enhanced robustness and easier tip change. An older signal generator was replaced with a professional PC audio card to provide more stable signal and more convenient on-screen fine tuning, also at a reduced cost. The Labview-based controlling program has also been improved through multiple iterations.Ph