Automated Detection and Quantitation of Bacterial RNA by Using Electrical Microarrays

Abstract

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

Novel Microarray Design Strategy To Study Complex Bacterial Communities

“…This target is particularly adapted to phylogenic studies since it contains highly conserved and variable moieties permitting reliable and detailed bacterial classification (12,25,37). In this approach, nucleic acids are directly extracted from samples without any prior cultivation; amplification is then performed using universal primers targeting conserved stretches of the 16S rRNA gene, and identification is based on similarity with sequences deposited in public ribosomal gene databases.…”

Discerning Trends in Multiplex Immunoassay Technology with Potential for Resource-Limited Settings

“…An electrochemical technology has been developed at the Fraunhofer Institute, and a device has been commercialized by eBiochip and referred to as the eMicro-ELISA (45 ). The device has been used for both multiplex nucleic acid detection and immunoassays, but all applications include an enzyme-labeled antibodybinding step.…”

“…For example, Quiel et al (46 ) have described its use for detection of bacterial toxins. The device consists of 16 electrode-array positions, each electrode array being made up of interdigitated gold elements (45 ). Capture antibodies are located on the electrodes and an automated ELISA is programed into the device.…”

Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria

“…The chip arrays are usually stored under nitrogen gas after their functionalization with capture probes [13]. Therefore, effects of chip array surface rehydration conditions on signal level were studied.…”