Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer spoilage bacteria Lactobacillus brevis.
Attomole (10(-18)mol) levels of RNA and DNA isolated from beer spoilage bacterial cells Lactobacillus brevis have been detected by the electrochemical sandwich DNA hybridization assay exploiting enzymatic activity of lipase. DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design. The assay employs magnetic beads (MB) modified with a capture DNA sequence and a reporter DNA probe labeled with the enzyme, both made to be highly specific for L. brevis DNA. Lipase-labeled DNAs captured on MBs in the sandwich assay were collected on gold electrodes modified with a ferrocene (Fc)-terminated SAM formed by aliphatic esters. Lipase hydrolysis of the ester bond released a fraction of the Fc redox active groups from the electrode surface, decreasing the electrochemical signal from the surface-confined Fc. The assay, shown to be efficient for analysis of short synthetic DNA sequences, was ineffective with genomic double stranded bacterial DNA, but it allowed down to 16 amole detection of 1563 nts long RNA, isolated from bacterial ribosomes without the need for PCR amplification, and single DNA strands produced from ribosomal RNA. No interference from E. coli RNA was registered. The assay allowed analysis of 400 L. brevis cells isolated from 1L of beer, which fits the "alarm signal" range (from 1 to 100 cells per 100mL).
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Ny Munkegade 1521, DK-8000 Aarhus C, Denmark.
Stepan
S
Shipovskov
Aaron M
AM
Saunders
Jesper S
JS
Nielsen
Majken H
MH
Hansen
Kurt V
KV
Gothelf
Elena E
EE
Ferapontova
D001515:Beer / Q000382:microbiology*; D015374:Biosensing Techniques / Q000379:methods*; D002175:Candida / Q000201:enzymology; D004269:DNA, Bacterial / Q000302:isolation & purification*; D055664:Electrochemical Techniques / Q000379:methods; D005656:Fungal Proteins / Q000378:metabolism; D052196:Levilactobacillus brevis / Q000302:isolation & purification*; D008049:Lipase / Q000378:metabolism; D008280:Magnetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D005656:Fungal Proteins; D012329:RNA, Bacterial; D008049:Lipase; C431349:lipase B, Candida antarctica