Highly rapid amplification-free and quantitative DNA imaging assay

Abstract

There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a 'sample-in-result-out' mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.

Figure 1. Principle of bead-based DNA quantification.

(a) Target DNA fragments are captured by beads functionalized with specific complementary oligonucleotides (capture probe) and labeled by reporter oligonucleotides with a fluorophore attached (detection probe). (b) Fluorescence of the beads is measured on a compact, robust and simplified wide-field fluorescence microscope. (c–e) Data analysis. (c) Beads are excited with different excitation intensities dependent on their position in the field of view. (d) Principles of data processing. The image is corrected for the variation in excitation across the image and then single beads are selected for analysis. (e) Dependency between mean bead intensity and position in the field of view. Raw and profile corrected intensity values.

Figure 2. Detection efficiency is controlled by microscope equipment and probe characteristics.

(a) Dependency of the detection sensitivity on microscope equipment. Target fragment denaturation and oligonucleotide hybridization was achieved by heating the sample for 2 minutes at 95°C with subsequent annealing to room temperature. Capturing of target DNA fragments was attained by incubating beads for 60 minutes at room temperature. High-end fluorescence microscope, miniaturized optical breadboard. (b) The substitution of DNA capturing probes by LNA modified strands increases the detection sensitivity. Dotted lines display the mean blank sample intensity level, the detection efficiency γ is defined as the ratio of specific to unspecific fluorescence signal. Error bars representing the standard error of mean bead intensities (SEM, n = 60–89 beads) of acquired images are smaller than data points.

Figure 3. Nonspecific adsorption of fluorophores (1 μM) on beads.

The fluorophores used in this study were obtained as N-hydroxysuccinimidyl esters. When dissolved in water, N-hydroxysuccinimidyl esters are hydrolyzed to carboxylic acids within a few hours. Beads exposed to Atto 647N, Cy5 and Alexa Fluor 647 were imaged with integrations times of 10, 100 and 100 ms, respectively. Error bars display the standard error of mean bead intensities (SEM) with n = 50.

Figure 4. Fluorescence intensity at the lowest detected target concentration.

Images obtained using (a) Atto 647N labeled detection probes (100 pM [target]) and (b) Alexa Fluor 647 labeled detection probes (10 pM [target]). First row: autofluorescence; second row: unspecific signal; third row: specific signal for 5 kbp target quantification. (i) Fluorescence images of beads. (ii) Expanded image of a selected bead. (iii) Fluorescence intensity profile plot of the bead shown in (ii). (iv) Mean fluorescence intensity and SEM of beads (n = 100).

Figure 5. Procedure scheme for DNA quantification.

(a) Overview of processing steps. (MPs: microparticles) (b) Oligonucleotide hybridization sequences. Blocking oligonucleotides (B1-5) assure the prevention of re-hybridization and thus the accessibility of capture probe (C) and detection probe (D) binding sequences on the target strand. (c) Sweep line. The whole assay procedure is accomplished in only three procedure steps in just 17 minutes processing time.

Figure 6. Quantification of 5 kbp DNA fragments.

(a) Comparison of detection efficiencies between differently labeled detection probes. Alexa Fluor 647, Atto 647N. (b) Mean fluorescence intensity values of 5 kbp DNA fragment using Alexa 647 labeled detection probes. Concentration of target fragments in spiked samples introduced in the assay, final concentration while incubation phase. (c) Visualization of increasing bead brightness with ascending target concentration using the Atto 647N label. Dotted lines display the mean blank sample intensity level; error bars represent the standard error of mean bead intensities (SEM, n = 53–97). Solid lines are guides to the eye. Error bars of (a) indicate the standard error of mean intensity (SEM) between three independent measurements demonstrating the reliability of the parameter.

Figure 7. Multiplexing.

Demonstration of multiplexed detection of fluorescently labeled beads by spectrally splitting the image into two channels: green, centered at 535 nm (a) and red, centered at 620 nm (b). Two fluorophores were used to label Dynabeads with a diameter of 2.8 μm: Atto 488 (emission maximum at 523 nm) and Atto Rh6G (emission maximum at 560 nm). Four types of beads are present in the images: unlabeled beads and three color codes: with Atto 488 only, with Atto Rh6G only and with a mixture of the two fluorophores at 1:5 ratio. (c) False color image displaying the logarithm r of the green-to-red ratio, clearly identifying all three types of labeled beads and the weakly autofluorescent unlabeled beads.