Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance

Abstract

The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

Fluorescent Probes of DNA Repair

“…To simplify workflow and allow for rapid separation of the probe from complex matrices, several groups have reported on-bead versions of molecular beacon repair probes. − Such probes operate either by generating a fluorescent on-bead light up or by releasing a fluorophore from the bead surface into solution. In one recent example, an on-bead probe of APE 1 was used in cells to explore APE 1 repair activity in tumors …”

Bioresponsive Release System for Visual Fluorescence Detection of Carcinoembryonic Antigen from Mesoporous Silica Nanocontainers Mediated Optical Color on Quantum Dot-Enzyme-Impregnated Paper

“…22,23 Porousness of paper makes it immobilize effectively and easily diffuse. Numerous researchers have attempted to develop nanometer-materials-based PADs (e.g., using electrochemiluminescence, 24 chemiluminescence, 25 fluorescence, 26,27 colorimetry 28 and electrochemistry 29,30 ) for application in the detection of ions, protein, DNA, and enzymes. Krull and co-workers 31 DNA hybridization assay.…”

Recent Advances and Applications in Paper-Based Devices for Point-of-Care Testing

“…Besides, enzyme activity also can be detected by μPads [ 179 – 181 ]. A λ exonuclease-assisted paper-based FL assay [ 179 ] was described for facile testing of polynucleotide kinase (PNK) activity by fluorescence intensity on paper surfaces, achieving sensitivity of PNK activity down to 0.0001 U/mL. In addition, paper-based FL immunoassays were used commonly for the detection of antibody biomarker [ 182 – 185 ].…”

“…Besides, μPads have measured various proteins (enzymes and antibodies) in whole blood, urine, saliva and sweat, as they are regarded as biomarkers of some diseases [176][177][178][179][180][181]. Lin's group has described a paper-based immunoassay for the matrix metalloproteinase-7 (MMP7) detection, which was corresponding to renal cancer [177].…”

“…Besides, enzyme activity also can be detected by μPads [179][180][181]. A λ exonuclease-assisted paper-based FL assay [179] was described for facile testing of polynucleotide Fig. 4 Some examples for FL detection.…”