Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria.
Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600mM sodium chloride and 40mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals up to twofold for longer DNA strands (i.e., >300bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease of recognition as follows: triple random>quintuple middle>triple middle>single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker.
Liu
Yanling
Y
School of Biotechnology, Royal Institute of Technology (KTH), S-10691 Stockholm, Sweden.
Elsholz
Bruno
B
Enfors
Sven-Olof
SO
Gabig-Ciminska
Magdalena
M
D001409:Bacillus cereus / Q000737:chemistry / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D020137:Base Pair Mismatch; D015374:Biosensing Techniques; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
D001427:Bacterial Toxins; D015342:DNA Probes; D004269:DNA, Bacterial