Tentacle probes: eliminating false positives without sacrificing sensitivity.
The majority of efforts to increase specificity or sensitivity in biosensors result in trade-offs with little to no gain in overall accuracy. This is because a biosensor cannot be more accurate than the affinity interaction it is based on. Accordingly, we have developed a new class of reagents based on mathematical principles of cooperativity to enhance the accuracy of the affinity interaction. Tentacle probes (TPs) have a hairpin structure similar to molecular beacons (MBs) for enhanced specificity, but are modified by the addition of a capture probe for increased kinetics and affinity. They produce kinetic rate constants up to 200-fold faster than MB with corresponding stem strengths. Concentration-independent specificity was observed with no false positives at up to 1 mM concentrations of variant analyte. In contrast, MBs were concentration dependent and experienced false positives above 3.88 muM of variant analyte. The fast kinetics of this label-free reagent may prove important for extraction efficiency, hence sensitivity and detection time, in microfluidic assays. The concentration-independent specificity of TPs may prove extremely useful in assays where starting concentrations and purities are unknown as would be the case in bioterror or clinical point of care diagnostics.
Satterfield
Brent C
BC
Harrington Department of Bioengineering, Arizona State University Tempe, AZ, USA.
West
Jay A A
JA
Caplan
Michael R
MR
D015374:Biosensing Techniques; D005189:False Positive Reactions; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D025202:Molecular Diagnostic Techniques; D015345:Oligonucleotide Probes / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D013816:Thermodynamics
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes