A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology

AbstractRapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3–300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstractThe colorimetric assay system for analyzing target oligonucleotides

[1]  Mingli Chen,et al.  A FRET ratiometric fluorescence sensing system for mercury detection and intracellular colorimetric imaging in live Hela cells. , 2013, Biosensors & bioelectronics.

[2]  C. Croce,et al.  Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Michael Rehli,et al.  miRNA expression profiling in melanocytes and melanoma cell lines reveals miRNAs associated with formation and progression of malignant melanoma. , 2009, The Journal of investigative dermatology.

[4]  Bin Zhao,et al.  Effects of 2'-O-methyl nucleotide on ligation capability of T4 DNA ligase. , 2014, Acta biochimica et biophysica Sinica.

[5]  Longhua Tang,et al.  Toehold-initiated rolling circle amplification for visualizing individual microRNAs in situ in single cells. , 2014, Angewandte Chemie.

[6]  A. Bosserhoff,et al.  Integrin β3 expression is regulated by let-7a miRNA in malignant melanoma , 2008, Oncogene.

[7]  Wei Xu,et al.  Ultrasensitive colorimetric DNA detection using a combination of rolling circle amplification and nicking endonuclease-assisted nanoparticle amplification (NEANA). , 2012, Small.

[8]  M. Ali,et al.  Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids. , 2008, Angewandte Chemie.

[9]  Hailing Jin,et al.  Stand-alone rolling circle amplification combined with capillary electrophoresis for specific detection of small RNA. , 2009, Analytical chemistry.

[10]  Sebastian D. Mackowiak,et al.  Circular RNAs are a large class of animal RNAs with regulatory potency , 2013, Nature.

[11]  F. Slack,et al.  RAS Is Regulated by the let-7 MicroRNA Family , 2005, Cell.

[12]  Lei Wang,et al.  A novel label-free cascade amplification strategy based on dumbbell probe-mediated rolling circle amplification-responsive G-quadruplex formation for highly sensitive and selective detection of NAD+ or ATP. , 2013, Chemical communications.

[13]  Patrick S Doyle,et al.  Ultrasensitive multiplexed microRNA quantification on encoded gel microparticles using rolling circle amplification. , 2011, Analytical chemistry.

[14]  Bin Zhao,et al.  Discriminative identification of miRNA let-7 family members with high specificity and sensitivity using rolling circle amplification. , 2015, Acta biochimica et biophysica Sinica.

[15]  Weian Zhao,et al.  Colorimetric and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme. , 2012, Analytical chemistry.

[16]  Daniel Irimia,et al.  Ultrasensitive detection of low-abundance surface-marker protein using isothermal rolling circle amplification in a microfluidic nanoliter platform. , 2011, Small.

[17]  A. Osbourn,et al.  L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs). , 2001, Nucleic acids research.

[18]  H. Ghourchian,et al.  Colorimetric monitoring of rolling circle amplification for detection of H5N1 influenza virus using metal indicator. , 2015, Biosensors & bioelectronics.

[19]  M. Ali,et al.  Colorimetric sensing by using allosteric-DNAzyme-coupled rolling circle amplification and a peptide nucleic acid-organic dye probe. , 2009, Angewandte Chemie.

[20]  J. Storhoff,et al.  A DNA-based method for rationally assembling nanoparticles into macroscopic materials , 1996, Nature.

[21]  张春阳,et al.  Sensitive and Label-free DNA Methylation Detection by Ligation-Mediated Hyperbranched Rolling Circle Amplification , 2012 .

[22]  Daniel K. Bonner,et al.  Self-assembled RNA interference microsponges for efficient siRNA delivery. , 2012, Nature materials.

[23]  Caifeng Ding,et al.  Cascade signal amplification strategy for the detection of cancer cells by rolling circle amplification and nanoparticles tagging. , 2012, Chemical communications.

[24]  Y. Yatabe,et al.  Reduced Expression of the let-7 MicroRNAs in Human Lung Cancers in Association with Shortened Postoperative Survival , 2004, Cancer Research.

[25]  J. Kjems,et al.  Natural RNA circles function as efficient microRNA sponges , 2013, Nature.

[26]  Ronghua Yang,et al.  Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. , 2010, Analytical chemistry.

[27]  H. Ju,et al.  Highly sensitive and selective chemiluminescent imaging for DNA detection by ligation-mediated rolling circle amplified synthesis of DNAzyme. , 2013, Biosensors & bioelectronics.

[28]  Li-pei Zhang,et al.  Label-free colorimetric sensing of ascorbic acid based on Fenton reaction with unmodified gold nanoparticle probes and multiple molecular logic gates. , 2012, Analytica chimica acta.

[29]  Shusheng Zhang,et al.  A chemiluminescence imaging array for the detection of cancer cells by dual-aptamer recognition and bio-bar-code nanoprobe-based rolling circle amplification. , 2013, Chemical communications.