Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide.
Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD(+)), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5' end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3' end. Only in the presence of NAD(+), the probe was linked at 5' and 3' ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM-500 nM with a detection limit of as low as 1 pM NAD(+), and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD(+) level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD(+), taking advantage of specificity, sensitivity, cost-efficiency, and simplicity.
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China.
Zhijuan
Z
Cao
Pei
P
Wang
Xue
X
Qiu
Choiwan
C
Lau
Jianzhong
J
Lu
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D018938:Caco-2 Cells; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D011088:DNA Ligases / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D051379:Mice; D009243:NAD / Q000032:analysis*; D049329:Nanostructures / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012834:Silver / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D015342:DNA Probes; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009243:NAD; D012834:Silver; D011088:DNA Ligases