Selection, Characterization, and Biosensing Application of High Affinity Congener-Specific Microcystin-Targeting Aptamers

Abstract

The efficiency of current microcystin detection methods has been hampered by the low detection limits required in drinking water and that routine detection is restricted to a few of the congeners with high degree of undesired cross-reactivity. Here, we report the development of novel microcystin-targeting molecules and their application in microcystin detection. We have selected DNA aptamers from a diverse random library that exhibit high affinity and specificity to microcystin-LR, -YR, and -LA. We obtained aptamers that bind to all chosen congeners with high affinity with K(D) ranging from 28 to 60 nM. More importantly, we also obtained aptamers that are selective among the different congeners, with selectivity from 3-folds difference in binding affinity to total discrimination (K(D) of 50 nM versus nonspecific binding). Electrochemical aptasensors constructed with the selected aptamers were able to achieve sensitive and congener-specific microcystin detection with detection limit as low as 10 pM.

Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor

“…As of now however, commercial ELISA and PP2A assays have yet to be validated for BGA supplements, and they face potential challenges with antibody and protein phosphatase specificity. Non-traditional screening methods for MC detection are being developed including a microcystin-targeting aptamer used in an electrochemical aptasensor (Ng et al, 2012). Additionally, drop-coating deposition Raman spectroscopy with spectral evaluation using principal component analysis (PCA) or peakheight ratio identification has been investigated (Halvorson et al, 2011).…”

DNA aptamers selection and characterization for development of label-free impedimetric aptasensor for neurotoxin anatoxin-a

“…For this, random ssDNA oligonucleotide library was designed according to previously reported protocol (Ng et al, 2012) in which it consists of a central randomized region of 60 nucleotides flanked by two constant primer-hybridization sites at the 3′ and 5′ primes (5′-ATACCAG CTTATTCAATT-N 60 -AGATAGTAAGTGCAATCT-3′). The primers were modified with fluorescein and a PEG linker followed by a poly-A tail as reported previously (Stoltenburg et al, 2005).…”

“…In a counter selection step, the DNA pool was incubated with negative beads (i.e., beads without ATX); followed by collection of DNA washings and then incubated with ATX-beads after the same heating and cooling treatment as normal selection round. Subsequently, the selected DNA pools were amplified by PCR, as previously reported (Ng et al, 2012). PCR products were dried by SpeedVac and resuspended in (50:50 v/v) water and formamide and heated to 55°C for 5 min, and finally loaded into 12% denaturing PAGE to separate the fluorescein labeled DNA strand from the double stranded PCR product.…”

Pulsed galvanostatic control of a solid-contact ion-selective electrode for potentiometric biosensing of microcystin-LR

“…However, the tedious procedures for the preparation of the antibodies may limit their wide applications. Recently, alternative biosensors using aptamers as bioreceptors have been proposed [15,16]. Unfortunately, the selected aptamers are available only to a few microcystin congeners.…”