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Ultrasensitive SPR detection of miRNA-93 using antibody-enhanced and enzymatic signal amplification

: Schmieder, Stefan; Weißpflog, Janek; Danz, Norbert; Hübner, Max; Kreth, Simone; Klotzbach, Udo; Sonntag, Frank


Engineering in life sciences 17 (2017), Nr.12, S.1264-1270
ISSN: 1618-2863
ISSN: 1618-0240
Bundesministerium für Bildung und Forschung BMBF
13N12848; DEMOST
Fraunhofer IWS ()
Fraunhofer IOF ()
biochip; miRNA; molecular diagnostic; surface plasmon resonance (SPR)

MiRNAs are endogenous non-coding RNA molecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present a SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10 pmol/l was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1 fmol/l.