DOI: 10.5061/dryad.k6djh9whk
title: DNA origami signal amplification in lateral flow immunoassays

description: Lateral flow immunoassays (LFIAs) enable a rapid detection of analytes in
 a simple, paper-based test format. Despite their multiple advantages, such
 as low cost and ease of use, their low sensitivity compared to
 laboratory-based testing limits their use in e.g. many critical
 point-of-care applications. Here, we present a DNA origami-based signal
 amplification technology for LFIAs. DNA origami is used as a structurally
 precise adapter to connect detection antibodies to tailored numbers of
 signal-generating labels. As a proof of concept, we apply the DNA origami
 signal amplification in a sandwich-based LFIA for the detection of cardiac
 troponin I (cTnI) in human serum. We show a 55-fold improvement of the
 assay sensitivity with 40 nm gold nanoparticle labels and an adjustable
 signal amplification of up to 125-fold with fluorescent dyes. The
 technology is compatible with a wide range of existing analytes, labels,
 and sample matrices, and presents a modular approach for improving the
 sensitivity and reliability of lateral flow testing.
description: # **DNA Origami Signal Amplification in Lateral Flow Immunoassays**
 [https://doi.org/10.5061/dryad.k6djh9wh](https://doi.org/10.5061/dryad.k6djh9wh) This folder contains the dataset supporting the findings of our research paper, i.e., the data for all figures presented in the manuscript. We have used transmission electron microscopy (TEM) imaging and agarose gel electrophoresis (AGE) for characterizing DNA origami nanostructures as well as DNA origami nanostructures combined with antibodies, gold nanoparticles, and fluorescent dyes. The DNA origami nanostructures have then been applied in various types of lateral flow immunoassays (LFIAs). The results of the LFIAs are presented as images of assay strips, including conventional photographs and fluorescence images, as well as numerical data of test line intensity values from image analysis. ## Description of the data and file structure The data provided in this folder includes both raw and processed data. The presented raw data includes original TEM images, gel scans, and lateral flow strip images. Processed, numerical data from the image analysis of lateral flow strip images is included for reproducing the scatter and line graphs presented in the paper (Figures 3 & 4). Additionally, we provide processed versions of some lateral flow strip images, where processing was carried out for enhancing the visual clarity of images presented in the paper. **Data formats** * Image Files .tif, .jpeg, and .png TEM images, gel scans, and raw and processed lateral flow strip images The titles of the files describe their type. If the images have been processed after acquisition, the processing is indicated in the file name. For example, "… contrast adjusted.tif" means that the brightness and contrast of the image has been adjusted. * Excel files .xlsx Used for organizing and presenting numerical data and unprocessed image files according to the Figure numbers used in the research paper. **File structure** The files are divided into three folders; Figure 2, Figure 3, and Figure 4, each of which is further divided into subfolders corresponding to figure panels (Fig 2b, Fig 2c, ...). The file structure thus corresponds to the order in which the data are presented in the figures of the research paper. In the list below, the file path and details of each data file are described separately. **Figure 2** Overview: TEM and AGE data on the characterization of DNA origami signal amplification structures and their complexation with antibodies, gold nanoparticles, and fluorescent dyes. Files in Subfolder “Fig 2b”: * H240215_H107.tif Original TEM image of a 6-helix bundle (6HB) DNA origami signal amplification structure combined with detection antibodies. Files in Subfolder “Fig 2c”: * 20230217_S+au_03.tif Original TEM image of a 6-helix bundle (6HB) DNA origami signal amplification structure combined with DNA-gold labels. * 2023-08-09 AGE 6HB A647-DNA excess screening Alexa647.tif Original, unprocessed image of an agarose gel imaged using 625/30 nm excitation and 695/55 nm detection, showing the fluorescence of Alexa Fluor 647 dyes on DNA origami structures. * 2023-08-09 AGE 6HB A647-DNA excess screening SYBR Safe.tif Original, unprocessed image of an agarose gel imaged under UV light, showing the fluorescence of Sybr Safe DNA intercalating dye in DNA origami structures. **Figure 3** Overview: data on the LFIA detection of a biotinylated DNA oligonucleotide target using a DNA origami signal amplification structure. Files in Folder “Figure 3”: * Fig3_data.xlsx * The sheet “Fig 3a” contains 5 unprocessed lateral flow test fluorescence images, imaged using 625/30 nm excitation and 695/55 nm detection, showing the fluorescence of Alexa Fluor 647 dyes on DNA origami structures. Each image is labeled with the number of Alexa Fluor 647-labeled DNA oligonucleotides used per one DNA origami structure, e.g., “6HB + 200x A647-DNA” refers to 6HB DNA origami with a 200-fold molar excess of Alexa Fluor 647-DNA oligonucleotides. Next to each image is a table describing the concentration of biotinylated DNA analyte, *c*(bt-DNA), on the test strips in the image. * The sheet “Fig 3b” contains test line intensity values extracted from the images in Sheet “Fig 3a” using an ImageJ analysis. * The sheet “Fig 3c” contains test line intensity values extracted from the images in Subfolder “Fig 3c processed images”. The data are grouped into three tables. The first table contains test line intensities from three repeated experiments. The names in column “Sample type” are structured as “Sample matrix, bt-DNA concentration, molar excess of Alexa Fluor 647-DNA oligonucleotides per DNA origami structure”, e.g., “Milli-Q H2O, 0 nmol/L, 200x”. The second table contains average values from Table 1, and the third table contains the standard deviation values. Files in Subfolder “Fig 3a-b processed images” * 24-05-16 Combined - 100x-unamplified-200x-50x-100xSerum - fire LUT - contrast adjusted.tif; 24-05-16 Combined - 100x-unamplified-200x-50x-100xSerum - fire LUT.tif; 24-05-16 Combined - 100x-unamplified-200x-50x-100xSerum.tif * o Processed images from the original images presented in the Sheet “Fig 3a” in the file Fig3_data.xlsx. The processing steps that have been done are indicated in the file names: cropping and combining multiple image files into a single file (“Combined”), applying fire LUT (“fire LUT”), and adjusting brightness/contrast (“contrast adjusted”). Files in Subfolder “Fig 3c processed images” * 2024-06-11_H2O_strips.tif; 2024-06-11_saliva_strips.tif; 2024-06-11_serum_strips.tif * o Cropped and combined, but otherwise unprocessed fluorescence images of LFIA tests used for acquiring the numerical data in the Sheet “Fig 3a” in the file Fig3_data.xlsx. **Figure 4** Overview: data on the LFIA detection of cardiac troponin I using a DNA origami signal amplification structure. Files in Folder “Figure 4” * Fig4_data.xlsx * o Sheet “Fig 4a” contains test line intensity values extracted from LIFA strip fluorescence images with ImageJ image analysis. Column 1 shows the concentration of cardiac troponin I in the sample in nanomoles per liter. Column 2 contains test line intensity values from the image “2024_02_22 Y302 6HB Alexa647 dilution series 15 min 16ms exposure.tif” stored in Subfolder “Fig 4a Fluorescence LFA troponin dilution series”. Column 3 contains the intensity values from image “2024_02_22 Y302 Alexa647 oligo dilution series 15min 16ms exposure.tif” in Subfolder “Fig 4a Fluorescence LFA troponin dilution series”. * o Sheet “Fig 4c” contains a table with test line intensity values extracted with ImageJ image analysis from LFIA strip photographs. Columns from left to right are: concentration of cardiac troponin IC complex (c(cTnIC)) in a sample in picomoles per liter; concentration of cardiac troponin I in a sample in nanograms per liter calculated from the values in column 1 and the molecular weight of cardiac troponin I in cell H9; test line intensity values from image file “2024_03_15 19C7cc 6HB NP dilution series 15min.JPG” in Subfolder “Fig 4b-c T19-gold LFA troponin dilution series”; test line intensity values from image file “2024_02_06 Y302 6HB NP dilution series 15min.JPG” in Subfolder “Fig 4b-c T19-gold LFA troponin dilution series”; test line intensity values from image file “2024_03_15 19C7cc state of the art NP dilution series 15min.jpg” in Subfolder “Fig 4b-c T19-gold LFA troponin dilution series”. * o Sheet “Fig 4d-e” contains, in the top panel, image analysis results of the image file “24-02-08 montage all repeats – annotated.tif“ in Subfolder “Fig 4d-e T19-gold LFA LoD determination“. The middle panel of tables contains fit details for linear regression (OriginPro 2023 version 10.0.0.154) to the data in the top panel. The bottom row of each table, colored green, contains the result of the calculation of limit of detection (LoD) values based on the fit. The bottom panel table contains the LoD values rearranged into a single table with mean and standard deviation for reproducing the graph in Fig. 4e of the paper. Files in Subfolder “Fig 4a Fluorescence LFA troponin dilution series” * 2024_02_22 All strips 15min 16ms exposure fire LUT.png; 2024_02_22 All strips 15min 16ms exposure.tiff; 2024_02_22 All strips 15min 40ms exposure contrast adjusted fire LUT.png; 2024_02_22 All strips 15min 40ms exposure fire LUT.png; 2024_02_22 Y302 6HB Alexa647 dilution series 15 min 16ms exposure.tif; 2024_02_22 Y302 Alexa647 oligo dilution series 15 min 1s exposure fire LUT contrast adjusted.png; 2024_02_22 Y302 Alexa647 oligo dilution series 15 min 1s exposure.tif; 2024_02_22 Y302 Alexa647 oligo dilution series 15min 16ms exposure.tif * o Unprocessed and processed fluorescence images of LFIA strips. The assay time (minutes) and th exposure time (in milliseconds) used in imaging are indicated in the file names. For processed images, the type of processing is indicated in the file name: “All strips” refers to cropping and combining images, "contrast adjusted” refers to adjustment of brightness and contrast, “fire LUT” refers to applying fire LUT. Files in Subfolder “Fig 4b-c T19-gold LFA troponin dilution series” * 2024_02_06 Y302 6HB NP dilution series 15min.JPG; 2024_03_15 19C7cc 6HB NP dilution series 15min.JPG; 2024_03_15 19C7cc state of the art NP dilution series 15min.jpg * o Unprocessed LFIA test strip photographs. * Subfolder “Processed” contains further versions of the abovementioned image files, processed by adjusting the brightness and contrast of the images, cropping the images, or combining the images with each other. Files in Subfolder “Fig 4d-e T19-gold LFA LoD determination“ * 24-02-08 montage all repeats – annotated.tif * o Cropped and combined, but otherwise unprocessed LFIA test strip grayscale photographs. The images are arranged into an 3 x 20 array where the three columns are three repeats of the same experiment and the 20 rows are the assay represent different assay times (1-20 min). In each image in the array, 8 test strips are shown, with different concentrations of cardiac troponin IC complex (cTnIC) in picomoles per liter, as indicated in the legend above the array.  ## Sharing/Access information A previous version of the article has been published on bioRxiv:  [https://doi.org/10.1101/2024.07.05.602214](https://doi.org/10.1101/2024.07.05.602214) The research article, published in Nature Communications, is open access.