Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes

Abstract

Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis. One of the major challenges in multiplexed analysis is to identify each specific reaction with a distinct label or 'code'. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.

Figure 1. Synthesis of nanobarcodes.

(a) Schematic illustration of synthesis of a typical Y-DNA-based nanobarcode building block. Three starting oligonucleotide components were partially complementary to each other as indicated in the drawing. One oligonucleotide possessed a sticky end, another one was labeled with a fluorescent dye and the third one was labeled with a fluorescent dye or a probe depending on the experimental design. (b) Schematic illustration of the construction of a typical DL-DNA-based nanobarcode. The nanobarcode building blocks were covalently linked with each other through complementary sticky-end ligations. (c) Schematic illustration of barcode decoding. The nanobarcodes 4G1R, 2G1R, 1G1R, 1G2R and 1G4R were decoded based on the ratio of fluorescence intensity. A molecular recognition element, a probe, was also attached to each nanobarcode. The resultant nanobarcodes possess not only coding capability and capacity, but also molecular sensing ability. With a preassigned code library (see Supplementary Table 5 online), the nanobarcodes could be used for molecular detection. (d) The real color of nanobarcodes in an agarose gel illuminated with a strong UV light. Lanes 1 and 7 are Alexa Fluor 488–labeled starting oligonucleotide component and Bodipy 630/650–labeled starting oligonucleotide component, respectively. Lanes 2, 3, 4, 5, 6 are nano-barcodes 4G1R, 2G1R, 1G1R, 1G2R and 1G4R, respectively.

Figure 2. Microbead-based DNA detection using fluorescence microscopy.

(a) Schematic drawing of a sandwiched DNA nanobarcode whose signal is amplified from polystyrene microbeads. Briefly, biotin-labeled capture probes were attached to avidin-functionalized polystyrene microbeads. Each batch of microbeads had only one type of capture probe before all batches were pooled together. DNA targets (that is, control or unknown samples) were then captured by specific microbeads first. Each report probe, which was linked to a particular nanobarcode, was designed to be complementary to another part of a specific target DNA and thus was able to be hybridized onto a specific microbead. Since each microbead bound a large amount of sandwiched complexes (that is, capture probes/target DNA/report probes/nanobarcodes), fluorescence signals were amplified. (b) Merged fluorescent colors (pseudocolors) of nanobarcodes from individual microbeads. (c) Multiple target detection (a total of four targets) was achieved via a two-colored fluorescence microscope using DNA nanobarcodes and microbeads. All scale bars, 5 μm.

Figure 3. DNA blotting assay with nanobarcodes.

(a) Schematic drawing of a dot-blotting detection of multiple DNA targets with nanobarcodes. Target DNA molecules were manually blotted onto a nylon-membrane. After prehybridization and blocking, a library of nanobarcode mixture was loaded onto the membrane. Through specific hybridizations with report probes that were functionalized with nanobarcodes, target DNA molecules were detected using a fluorescence reader, scanner or microscope. (b) DNA from multiple pathogens (four in total) were detected simultaneously using nanobarcodes. Control 1 was a 27-mer ssDNA with unrelated sequences and control 2 was a plasmid DNA, pVAX1/lacZ. Scale bar, 1 mm.

Figure 4. Multiplexed DNA detection using flow cytometry.

(a) A two-color flow plot of microbeads attached with the nanobarcode 2G1R as a control for standards (a calibration control). FL1H indicates the green channel and FL4H is the red channel. (b) Simultaneous detection of three pathogen DNA using nanobarcodes. Unrelated DNA sequences were not detected (background).