Article pubs.acs.org/ac
Visual Detection of DNA on Paper Chips Yajing Song,† Péter Gyarmati,† Ana Catarina Araújo,‡ Joakim Lundeberg,† Harry Brumer, III,‡,§ and Patrik L. Ståhl*,∥ †
Division of Gene Technology, School of Biotechnology, Science for Life Laboratory, Royal Institute of Technology (KTH), SE-171 65 Solna, Sweden ‡ Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-106 91 Stockholm, Sweden § Michael Smith Laboratories and Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver V167T 1Z4, Canada ∥ Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Solna, Sweden S Supporting Information *
ABSTRACT: On-site DNA analysis for diagnostic or forensic purposes is much anticipated in the future of molecular testing. Yet the challenges to achieve this goal remain large with rapid and inexpensive detection and visualization being key factors for any portable analysis system. We have developed a filter paper-based nucleic acid assay, which is able to identify and distinguish dog and human genomic and mitochondrial samples in a forensic setting. The filter paper material allows for transport by capillary force of the sample DNA through the detection surface, allowing the targets to hybridize specifically to their complementary capture sequences. Coupling micrometer-sized beads to DNA allows the results to be visualized by the naked eye, enabling instant, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumentation.
“bioactive paper”, that is, advanced, cellulose-based functional material that has the capacity to bind, detect, and/or (de-) activate biological substances, ranging from small molecules, proteins, carbohydrates, and nucleic acids to whole organisms such as fungi, bacteria, and viruses18−30 (and references therein). Composed of a matrix of cellulose fibers, paper has a porous structure, which greatly increases the reactive surface area compared to glass and plastic slide surfaces.31 Indeed, paperbased bioassay platforms date back to the early 20th century; the development of paper chromatography led to the Nobel Prize in chemistry in 1952. This was the first time that a paper strip was turned into a commercial product32 and has since been followed by highly impactful lateral flow devices such as the pregnancy test strip.33 Whereas the majority of paper-based diagnostic applications have focused on protein or small-molecule detection,20−22,24 there is currently a large potential to make use of paper substrates for the analysis of DNA in diverse field applications, in which simplicity, affordability, sensitivity, specificity, rapidity, and robustness are all essential. In response to these challenges, we recently devised a novel paper-based DNA array technology by combining new celluloseprobe coupling chemistry with rapid target hybridization driven
T
oday, nucleic acid assays are essential tools for the characterization of microorganisms, for genotyping, and for gene expression profiling. Their utility is continuously increasing in the areas of routine and on-site testing, where they are used for widespread applications such as characterization of infectious diseases, food and water testing, and in forensic science.1−6 To facilitate this evolution, the ease of use and high throughput of these molecular assays are essential.1,4 To date, assays have employed microarray technology as a high-throughput multiplex detection platform.7−12 Since the 1990s, two-dimensional hybridization microarrays have been developed on solid substrates, usually glass, plastic, or silicon.13,14 Three-dimensional suspension bead array technology is also widely used today, and early multiplex detection of nucleic acids has led to assays for MHC tissue typing and pathogen detection.15 In suspension bead arrays, hybridization kinetics is better than that on planar arrays;16 however, specific annealing temperatures are required in each system.17 The main drawbacks of these traditional multiplex detection systems in that they require expensive instrumentation for detection and extensive protocols for preparation of the detection surface and samples. For instance, fluorescent detection of a DNA probe on a microarray requires conjugating the DNA to a fluorescent molecule, as well as a fluorescent scanner to perform the actual assay readout.7−12 With an aim of developing and improving simpler diagnostic tests, increasing attention is focused on the development of © 2014 American Chemical Society
Received: October 4, 2013 Accepted: January 2, 2014 Published: January 2, 2014 1575
dx.doi.org/10.1021/ac403196b | Anal. Chem. 2014, 86, 1575−1582
Analytical Chemistry
Article
DNA, dog mitochondrial DNA and dog genomic DNA, respectively, were designed with a 5′ biotin-modified forward primer and a reverse primer incorporating one of the four capture-tag sequences (T_1-T_4) used in the immobilized capture-tag oligos. The region spanned by the primers also incorporated the internal sequence (N_1-N_4) used in the immobilized internal probe capture oligos (Figure 1, Table S2 of the Supporting Information).
by capillary transport through a paper matrix. In a practical example, a tetraplex assay was used to discriminate amplicons of canine and human DNA in a simulated forensic analysis, with the hybridization step occurring in
Visual Detection of DNA on Paper Chips Yajing Song,† Péter Gyarmati,† Ana Catarina Araújo,‡ Joakim Lundeberg,† Harry Brumer, III,‡,§ and Patrik L. Ståhl*,∥ †
Division of Gene Technology, School of Biotechnology, Science for Life Laboratory, Royal Institute of Technology (KTH), SE-171 65 Solna, Sweden ‡ Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE-106 91 Stockholm, Sweden § Michael Smith Laboratories and Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver V167T 1Z4, Canada ∥ Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Solna, Sweden S Supporting Information *
ABSTRACT: On-site DNA analysis for diagnostic or forensic purposes is much anticipated in the future of molecular testing. Yet the challenges to achieve this goal remain large with rapid and inexpensive detection and visualization being key factors for any portable analysis system. We have developed a filter paper-based nucleic acid assay, which is able to identify and distinguish dog and human genomic and mitochondrial samples in a forensic setting. The filter paper material allows for transport by capillary force of the sample DNA through the detection surface, allowing the targets to hybridize specifically to their complementary capture sequences. Coupling micrometer-sized beads to DNA allows the results to be visualized by the naked eye, enabling instant, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumentation.
“bioactive paper”, that is, advanced, cellulose-based functional material that has the capacity to bind, detect, and/or (de-) activate biological substances, ranging from small molecules, proteins, carbohydrates, and nucleic acids to whole organisms such as fungi, bacteria, and viruses18−30 (and references therein). Composed of a matrix of cellulose fibers, paper has a porous structure, which greatly increases the reactive surface area compared to glass and plastic slide surfaces.31 Indeed, paperbased bioassay platforms date back to the early 20th century; the development of paper chromatography led to the Nobel Prize in chemistry in 1952. This was the first time that a paper strip was turned into a commercial product32 and has since been followed by highly impactful lateral flow devices such as the pregnancy test strip.33 Whereas the majority of paper-based diagnostic applications have focused on protein or small-molecule detection,20−22,24 there is currently a large potential to make use of paper substrates for the analysis of DNA in diverse field applications, in which simplicity, affordability, sensitivity, specificity, rapidity, and robustness are all essential. In response to these challenges, we recently devised a novel paper-based DNA array technology by combining new celluloseprobe coupling chemistry with rapid target hybridization driven
T
oday, nucleic acid assays are essential tools for the characterization of microorganisms, for genotyping, and for gene expression profiling. Their utility is continuously increasing in the areas of routine and on-site testing, where they are used for widespread applications such as characterization of infectious diseases, food and water testing, and in forensic science.1−6 To facilitate this evolution, the ease of use and high throughput of these molecular assays are essential.1,4 To date, assays have employed microarray technology as a high-throughput multiplex detection platform.7−12 Since the 1990s, two-dimensional hybridization microarrays have been developed on solid substrates, usually glass, plastic, or silicon.13,14 Three-dimensional suspension bead array technology is also widely used today, and early multiplex detection of nucleic acids has led to assays for MHC tissue typing and pathogen detection.15 In suspension bead arrays, hybridization kinetics is better than that on planar arrays;16 however, specific annealing temperatures are required in each system.17 The main drawbacks of these traditional multiplex detection systems in that they require expensive instrumentation for detection and extensive protocols for preparation of the detection surface and samples. For instance, fluorescent detection of a DNA probe on a microarray requires conjugating the DNA to a fluorescent molecule, as well as a fluorescent scanner to perform the actual assay readout.7−12 With an aim of developing and improving simpler diagnostic tests, increasing attention is focused on the development of © 2014 American Chemical Society
Received: October 4, 2013 Accepted: January 2, 2014 Published: January 2, 2014 1575
dx.doi.org/10.1021/ac403196b | Anal. Chem. 2014, 86, 1575−1582
Analytical Chemistry
Article
DNA, dog mitochondrial DNA and dog genomic DNA, respectively, were designed with a 5′ biotin-modified forward primer and a reverse primer incorporating one of the four capture-tag sequences (T_1-T_4) used in the immobilized capture-tag oligos. The region spanned by the primers also incorporated the internal sequence (N_1-N_4) used in the immobilized internal probe capture oligos (Figure 1, Table S2 of the Supporting Information).
by capillary transport through a paper matrix. In a practical example, a tetraplex assay was used to discriminate amplicons of canine and human DNA in a simulated forensic analysis, with the hybridization step occurring in
