Rapid Amplification-Free Microarray-Based Ultrasensitive Detection of

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Rapid Amplification-Free Microarray-Based Ultrasensitive Detection of DNA Yuri M. Shlyapnikov, Ekaterina A Malakhova, and Elena Andreevna Shlyapnikova Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02149 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 30, 2019

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Analytical Chemistry

Rapid Amplification-Free Microarray-Based Ultrasensitive Detection of DNA Yuri M. Shlyapnikov*, Ekaterina A. Malakhova, and Elena A. Shlyapnikova Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia. *Correspondence. E-mail: [email protected] Fax: 7-496-733-0553. ABSTRACT We present a multiplex microarray-based assay of DNA fragments, which allows the detection of less than 10,000 DNA fragments in a sample of 100 µL (corresponding to ~0.1 fM analyte concentration) in less than 5 min. High speed and sensitivity are due to three main features of the assay. First, biotinylated adapter oligonucleotides are hybridized to the DNA fragment. Second, it is electrophoretically concentrated from the sample onto the microarray. Third, biotin labels are detected by scanning the microarray surface with streptavidin-coated magnetic beads. Prior to analysis, dsDNA fragments and genomic DNA samples were first denatured and then annealed in the presence of blocking oligonucleotides generating ssDNA fragments capable of hybridizing with oligonucleotide probes on the microarray. The multiplexity of the assay system was demonstrated by the simultaneous detection of the genomic DNAs of three microorganisms: E. coli, B. cereus, and M. neoaurum. Key words: ultrasensitive assay, hybridization analysis, DNA microarray, magnetic beads. Abbreviations: EDC, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; LOD, limit of detection; NHS, N-hydroxysuccinimide; PVP, polyvinylpyrrolidone. INTRODUCTION Polymerase chain reaction is now a well recognized gold standard in many analytical applications1. It is extremely sensitive, so that single DNA molecules can be detected2. However, the assay time cannot be substantially reduced from minimum limit of 30 min for 40 cycles3. Other limitations of PCR based methods include: (i) susceptibility to false negative results due to the presence of inhibitors in probes, (ii) the dependence of amplification efficiency on the DNA sequence, (iii) possible amplification errors and the formation of chimeric and heteroduplex molecules4, and limited multiplexing capability5. In the case of cDNA assay, additional errors may be introduced by the reverse transcriptase employed in analysis. PCR-based assays include an amplification of the target DNA sequence, and thus may be referred to as target amplification methods. At the expense of some loss in the assay limit of detection (LOD), PCR-free techniques have some benefits in that they accelerate the assay or reduce its sensitivity to the presence of ACS Paragon Plus Environment

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impurities in probes. Table 1 summarizes some published data on PCR-free techniques with an emphasis on rapid and highly sensitive methods. Most of them also use some kind of amplification, but the signal or probe are amplified instead of the target. Probe amplification methods are based on a ligase or polymerase reactions. They provide high specificity and are often used to detect point mutations, but the complex and long-lasting assay procedure limits their use6,7. Yet another way to increase the sensitivity is to amplify the signal. This approach is common for various electrochemical sensor systems where the single label generates multiple reaction events, which in their turn generate the signal. The electrochemical detection can be achieved without any labels, with dsDNA itself acting as a label while still including the signal amplification8, 9. Another way to amplify the signal is to grow gold particles on the labels which can be further detected7, 10-12. It should be noted that signal amplification usually entails either an increase in the duration of assay or the need for complex analytical procedures which often result in high cost of analysis. Table 1. Review of PCR-free hybridization assay techniques. The performance of the assay reported in the present work is shown for comparison. Method used

LOD,

Assay time

Reference

concentration or amount Electrochemical detectiona

1 fg/μL

30 min

8

Chemiluminescencea

0.3 fM

> 100 min

10

Electrochemical detectiona

0.6 fM

> 90 min

11

Single quantum dots analysisa

zeptomoles

1h

13

Surface plasmon resonance imaginga

10 fM

100 s

14

> 40 min

15

>3h

16

Сatalyzed gold deposition measured by 0.3 fM quartz-crystal-microbalancea Coulometric

measurement

of

gold 1 fM

nanoparticle-mediated electron transfera Single-molecule fluorescenceb

0.1 fM

> 10 min

17

Capillary electrophoresis,

0.3 fM

10 min +

18

single-molecule imagingb

electrophor esis time

Electromechanical signal transductionb

10 fM

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> 12 h

19

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Epifluorescence microscopy, single molecule 1.3 fM

>1h

20

detectionb Fluidic force discrimination assayb

9 fM

25 min

21

Electrophoresis, magnetic labelsb

0.1 fM or

5 min

Present work

10 zeptomoles a

– signal or probe amplification, b – amplification-free methods Thus, the majority of known ultrasensitive methods include some kind of amplification (of

target, probe or signal). Until now, no simple and rapid method has been known which was completely amplification-free and at the same time had a sensitivity at the level of a thousand molecules. As can be seen in Table 1, femtomolar sensitivity in the signal and probe amplificationfree assays was only achieved with single-molecule fluorescence detection which requires sophisticated and expensive optical devices22. In our previous publication23 we employed magnetic beads as labels sensitive to single hybridization events (this means that the label may be held on the surface with a single link between complementary DNA sequence). We were able to detect 3 amole of a biotinylated oligonucleotide in a 3 μL microdroplet probe within 2 hours. Somewhat higher assay characteristics were achieved by a similar approach, employing viscous drag force instead of magnetic field21, 24. Further decrease in the assay time and in LOD can be achieved by applying the “active” assay principle25. The technology combines two approaches, electrophoresis and force discrimination, which eliminate the diffusion on all assay stages. When applied to immunoassay, they allowed to determine 1000 molecules in 5 min, with the possibility of overcoming the crossreactivity of antibodies26. So far, this platform has been applied exclusively for the detection of protein molecules and viruses. The goal of the present work was to develop the DNA hybridization assay employing the “active” assay technology. We aimed at the creation of an analytical method, which would be rapid, simple, and at the same time highly sensitive and specific enough to work with the real biological samples of complex composition. We also studied the possibility of detection of single nucleotide mismatches using the force discrimination of DNA duplexes. EXPERIMENTAL SECTION Reagents. A regenerated cellulose membrane with a thickness of 40 μm (3.5 kDa cutoff) and all chemicals were purchased from Sigma-Aldrich Co. Carboxylic (Dynabeads MyOne) and streptavidin coated magnetic beads (Dynabeads MyOne C1), both 1 µm in diameter, were purchased ACS Paragon Plus Environment


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from ThermoFisher. Oligonucleotides were synthesized by the Synthol (Russia). Covalent immobilization of oligonucleotides on carboxylated magnetic beads was performed according to the manufacturer’s protocol. Human mitochondrial DNA and the cultures of E. coli, M. neoaurum, and B. cereus were kindly provided by Dr. T. V. Ivashina. The sequences of oligonucleotides used are given in Table S1 in the Supporting Information. Obtaining fragments of human mitochondrial DNA. Double-stranded fragments were obtained by the symmetric PCR, single-stranded fragments were obtained by the asymmetric PCR with an excess of the second primer in relation to 30:1. The resulting fragments were purified by electrophoresis in polyacrylamide gel with the photometric determination of concentration. Fabrication of DNA microarrays. The membrane surface was activated in a 1% solution of cyanuric chloride in dry toluene for 1 min (see Supporting Information). Electrospray deposition of oligonucleotides on the activated surface was carried out from 10 μM solutions containing 1% trehalose through a 250 μm polyester mesh mask according to our previous publication27. After the deposition of oligonucleotides, microarrays were incubated in a humid atmosphere for 30 min. Remaining active groups were quenched by a 0.5 M solution of ethanolamine, pH 9, for 1 h. Microarrays were rinsed with a 1% trehalose solution, air-dried, and stored at -20 °C. Samples of double-stranded DNA fragments for hybridization analysis were prepared in two ways: 1) Melting of double-stranded DNA by heating 100 μL of the sample to 100°C for 30 s after which the sample was immediately used for analysis. 2) Melting of double-stranded DNA by heating the sample to 100ºC for 30 s with an excess of blocking oligonucleotides with a concentration of each one equal to 1 μM. Analysis of DNA fragments. The design of a flow cell for an ultrasensitive microarray-based analysis is described in detail in the previous publications25, 27. The analysis of single-stranded DNA fragments was performed in two ways, as illustrated in Fig. 1. According to option 1, 100 μL of the sample in the “Im-Gly” buffer (containing 20 mM imidazole titrated with glycine to pH 8.5, as well as a 0.1% Tween-20 and a 1% PVP) was pumped at a rate of 30 μL /min through a flow cell with a microarray with an electric field (voltage 100 V, current 5-10 mA) applied. Then, the electric field was turned off, the conic magnet was placed under the flow cell, and a 0.001% suspension of magnetic particles coated with the detecting oligonucleotides was pumped through the cell in the Im-Gly buffer at a rate of 10 ACS Paragon Plus Environment

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μL/min for 30-60 s. According to option 2, immediately before the analysis, 10 μL of the solution of a biotinylated adapter oligonucleotide with a concentration of 10 μM were added to 100 μL of the fragment solution being analyzed. The sample obtained was analyzed as described above using streptavidin-coated magnetic beads. The image of the microarray was taken with a CCD camera and processed as described in the Supporting Information. Preparation of samples for analysis of bacterial pathogens. A suspension containing a known number of cells of microorganisms in an “Im-Gly” buffer, as well as a 0.1 nM fragment of human mitochondrial ssDNA used as a positive control, 3 μM of biotinylated adapter oligonucleotides, and a 1 μM of blocking oligonucleotides were sonicated for 30 s at 100% power (Cole Palmer Ultrasonic Processor). Then, the samples were incubated at 100 ºС for 30 s and immediately used for analysis. Safety Considerations. The electrophoretic unit should be placed on a nonconductive table and should not be touched while running electrophoresis. RESULTS AND DISCUSSION Hybridization analysis and determination of the limit of detection of a single-stranded DNA fragment. The most direct way to conduct the hybridization analysis of a non-labelled single-stranded DNA fragment is to use a pair of specific oligonucleotides, one as a probe on a microarray, and the other on the bead, as illustrated in Figure 1A. However, when the assay was performed by scanning the microarray with oligonucleotide-coated magnetic beads in a flow cell, no signal was observed even at an analyte concentration of 1 nM. By contrast, the “push-pull” assay (see Supporting Information) with the same microarray and the same analyte concentration produced a high signal. We suppose that the beads move too fast, and DNA duplexes do not have sufficient time to form. In contrast to the previously published work21, which uses an additional three-minutes long step of hybridization-mediated binding of beads to a sensor, here we use a more rapid technique, where the beads are constantly moving over the microarray surface. To overcome the kinetic limitations described, we applied a more complex technique employing biotinylated adapter oligonucleotides, as Figure 1B illustrates. Whereas DNA strands anneal with rate constant of 105-106 M-1s-1 at 0.5 M salt concentration29, 30 and even slower at low ionic strength conditions31, streptavidin-biotin complexes are formed very rapidly with a rate constant of 107 M-1s-1

32.

An additional advantage provided by the use of biotin labels is the

possibility to simultaneously detect several analytes using a single sort of beads, just the same as described earlier for the multiplex immunoassay27. ACS Paragon Plus Environment


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Figure 1. Two options for conducting “active” hybridization analysis. (A) DNA fragments being analyzed are electrophoretically captured on a microarray and then detected with magnetic beads coated with specific oligonucleotides. (B) Prior to the assay, DNA fragments are hybridized with specific biotinylated adapter oligonucleotides and then are collected on a microarray. Biotin labels are then revealed employing streptavidin-coated magnetic beads. With the use of biotinylated adapters, high sensitivity of the assay was achieved, as Figure 2 illustrates. In all experiments, the sample volume was 100 μL. As the volume increased to 1 mL, an increase in the signal intensity was observed corresponding to a tenfold increase in the DNA concentration. Using electrophoretic concentration, the detection limit for a ssDNA fragment with a length of 70 bases is 0.1 fM, which corresponds to 6000 analyte molecules in a sample. In this case the analysis time does not exceed 5 min. Examples of the microarray images for different concentrations of the model ssDNA fragment are presented in Figure 2A. For comparison, the results of hybridization analysis of the same DNA fragment without the use of electrophoresis are shown in Figure 2B. In this case, the LOD is 0.1 pM. Based on this value, we may calculate an efficiency of electrophoretic concentration of an analyte. In our experimental conditions, a piece of microarray containing about ten 100×100 µm2 spots is located in a flow cell with a width of 1.5 mm and a height of 100 μm. Following the model of diffusion-controlled delivery of analyte to the sensor surface33, we can conclude that ~104 molecules are captured on a microarray from a 100 fM solution in the absence of an electric field. As seen in Figure 2B, the signal value for an electrophoresis-free assay of 100 fM analyte solution corresponds to the electrophoresis-assisted assay of a 0.1 fM sample containing about 6000 analyte molecules. It can be assumed that the number of analyte molecules captured on the microarray is also almost the same. Thus, we can conclude that each analyte molecule in a sample is captured on a microarray in the electrophoresis-assisted assay.


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Figure 2. The results of hybridization analysis for different concentrations of the model single-stranded DNA fragment: (A) using an electrophoretic concentration of an analyte; (B) without electrophoretic concentration of an analyte. In each case, a representative fragment containing six active zones of the microarray surface is presented. The dependence of the signal intensity the average number of bound magnetic beads on a microarray spot on the concentration of a ssDNA fragment has a sigmoid shape with a dynamic range of about four orders of magnitude, and a saturation of signal is reached at an analyte concentration of ~1 pM (see Figure 3A). As seen in Fig. 2, some background binding of magnetic beads is observed at high analyte concentrations. At the same time, low analyte concentrations go along with low background, 0-3 beads per 100×100 μm2. Thus, such a low background does not affect the lower bound of the dynamic range. Another factor affecting the dynamic range is the nonuniform coverage of the microarray spot with magnetic beads at high analyte concentrations, which is usually observed in assays with magnetic beads scanning25. The maximum number of beads that a microarray spot can accommodate is never reached in such assays. Combined with the fact that beads cannot be accurately counted at high density, this leads to high variations of measured signal values and may reduce the dynamic range. Similar calibration curves were previously obtained for various immunochemical test systems with magnetic labels with the same dynamic range28. LOD values also coincide up to an order of magnitude. The similarity between hybridization and immunochemical assays suggests the generality of the factors governing the binding of magnetic beads to the microarray. These factors seem to be mainly the geometric ones, such as immobilization density of interacting molecules, their steric accessibility, and the surface roughness of a microarray and beads. At the same time, the nature of specific bonds, whether they are antigen-antibody or complementary nucleic acid interactions, does not play a significant role. The main difference seems to be the kinetic one: as



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discussed above, a DNA duplex needs more time to form compared to streptavidin-biotin and antigen-antibody complexes. Finally, it is possible to estimate the fraction of analyte molecules that are labeled with magnetic beads. As shown above, we may assume that when 100 μL of a 0.1 fM solution is analyzed, every analyte molecule is captured on the microarray. This corresponds to 600 DNA fragments per microarray spot, taking into account that ten repeats of microarray are present in the flow cell. About 20 magnetic beads are bound in each spot in this case, which means that only 1/30 of the analyte molecules are labeled, while the others are probably sterically hindered due to the irregularity of the microarray surface. Detection of double-stranded DNA fragments. Obviously, the detection of dsDNA fragments is of much greater practical interest compared to single-stranded. However, dsDNA cannot hybridize with probes on a microarray and, therefore, be detected. Therefore, in order to adapt the described method for the detection of dsDNA, an additional procedure aimed at preparing single-stranded fragments is required. Initially we assumed that it is enough to melt the DNA immediately before analysis. Presumably, due to the low concentration of DNA in the sample, the recombination of ssDNA would occur more slowly than their hybridization on a microarray. Indeed, considering the highest possible association rate constant of k ~107 M-1s-1, at femtomolar DNA concentration the characteristic time needed for complementary strands to anneal is t > 1/kc = 108 s. However, when analyzing dsDNA with its preliminary melting, a significant decrease in signal intensity was observed, as well as an increase in the detection limit by an order of magnitude to 1 fM (see Figure 3A). Probably, the melted strains are not uniformly distributed over the sample volume but remain sterically close to each other, so that they are able to anneal back before being captured on a microarray. In this regard, we employed a more complex sample preparation protocol based on the melting of dsDNA analyzed in the presence of a set of blocking oligonucleotides, as illustrated in Figure 3B. The sequences of blocking oligonucleotides were designed to hybridize with the chains of melted DNA, preventing them from being annealed back, and, at the same time, not to affect hybridization with oligonucleotide probes on the microarray21. In the case of the addition of blocking oligonucleotides to the sample before melting, the detection limit for the model 713 bp long dsDNA fragment was 0.1 fM (see Figure 3A). The size of the dsDNA fragment was close to the size of the genomic DNA fragments obtained by sonication, as discussed below. The data shown in Figure 3A suggest that blocking oligonucleotides completely protect the analyzed region of the DNA sequence from annealing to the complementary strand. Thus, the method of "active" hybridization analysis was adapted for the detection of dsDNA fragments without any loss of sensitivity. At the same time, an additional stage of DNA melting and


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annealing of blocking oligonucleotides takes no more than one minute and does not significantly affect the overall assay time.

Figure 3. (A) The dependence of signal on the concentration of ssDNA (blue curve) and dsDNA fragments (green curve - in the presence of blocking oligonucleotides, purple - without blocking oligonucleotides). The background level was 4 ± 3 beads/spot. Error bars correspond to 2.5×STD. (B) General strategy for the detection of dsDNA fragments. The complementary strands are melted, and blocking oligonucleotides are annealed on one of the strands, while the specific regions of the opposite strand become available for hybridizing with an adapter oligonucleotide and a microarray probe. Application of "active" hybridization analysis for multiplex determination of E. coli, B. cereus and M. neoaurum by specific DNA markers. Finally, we demonstrate a direct multiplex detection of bacterial genomic DNA in the cell culture. The microorganisms used in our model are M. neoaurum, which is highly similar to tuberculosis pathogen, B. cereus, which can serve as a model of the anthrax pathogen, and E. coli. Short (~1 kbp) fragments of genomic DNA were generated by sonicating bacterial cells as described previously21. The design of a microarray for the multiplex determination of three microorganisms is presented in Figure 4A. It was shown that the detection limit for each of the microorganisms is ~104 cells in a sample volume of 100 μL (Figure 4B). Assuming that each cell contains a single copy of the gene, this corresponds to a sensitivity of ~ 0.1 fM, which coincides with the sensitivity estimate given above for dsDNA. Thus, the presence in the sample of interfering substances, such as denatured proteins, fragments of biological membranes and cell walls, and a large excess of DNA ACS Paragon Plus Environment


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and RNA fragments, does not affect the sensitivity of the analysis. Figure S2 demonstrates the high specificity of the assay, where no false positive signals are observed at a concentration of ~ 107 cells /100 µL. The outstanding specificity of force discrimination assays has been previously demonstrated for protein immunoassay, where ~5×1010 molar excess of non-target proteins reduced the signal only by 10-15%25. Here we demonstrated that force discrimination-based hybridization assay is also highly specific: the huge excess of interfering bacterial DNA and RNA fragments does not affect the LOD. Assuming that each bacterial cell contains ~ 10-7 µg of nucleic acids34, we may conclude that the assay detects a single target sequence in the presence of ~108 bp of non-target sequences. We tried to use the high specificity of force discrimination to detect singe-base mismatches. However, as discussed in the Supporting Information, poor results were obtained. It should be also noted that high assay specificity is determined not only by the sequences of a probe and an adapter, but by the sequences of blocking oligonucleotides as well. Finally, we must note the high reproducibility of the assay. The LOD values for both model DNA fragments (~0.1 fM), and genomic DNA (~103 cells per sample) were confirmed in 5 independent experiments each. Since many copies of a microarray are accommodated in the flow cell during the analysis, each microarray spot is present in ≥ 10 repeats in the assay. This greatly reduces the possible signal variations. Although each bead-microarray contact is subject to large heterogeneities of both interacting surfaces, the averaging over a large number of bead-microarray contacts eliminates these variations. This makes the whole assay highly reproducible.

Figure 4. (A) The design of a microarray for the simultaneous detection of three microorganisms. “+K” denotes positive control (a spot of an oligonucleotide complementary to a human mitochondrial ssDNA fragment, which was added to each sample at 0.1 nM concentration). “-K” denotes negative control (a spot of a non-specific oligonucleotide). The results of a hybridization analysis in a sample, containing (B) ~ 103, (C) ~ 104 and (D) ~ 105 cells of each of the microorganisms. The sample volume was 100 μL. Representative fragments of the microarray surface are presented. CONCLUSIONS ACS Paragon Plus Environment

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The development of a fundamentally new method of "active" hybridization analysis, which allows detecting up to 6000 DNA molecules in 100 μL of solution (0.1 fM) in 5 min, is the main result of this work. No target, signal or probe amplification are used to enhance the method sensitivity. Instead, the ultra-high sensitivity and speed of the method are provided by the stages carried out by the action of a directed force: electrophoretic concentration of the analyzed DNA fragments and the magnetic field-assisted detection of analyte molecules. The method does not require large material investments; besides the flow cell of a rather sophisticated design, only inexpensive equipment is needed. Although the detection limit of the technique is higher than that of PCR-based methods, it has several significant advantages such as speed and high multiplexity limited only by the number of probes on the microarray. We believe that it could be of practical interest in the analytical applications requiring a quick response, for example, in medical diagnostics. The method can also be used to study DNA nanomechanics, since it provides a new way of loading DNA duplexes. In the traditional atomic force spectroscopy, the force of duplex rupture is measured at a given loading rate35. Instead, with a shear flow acting on the surfacetethered beads, one can measure the rate of dissociation of DNA strands under the constant action of the controlled force. Yet another option enabled by the technique developed is DNA hybridization and dehybridization controlled by the action of electrostatic force on the DNA fragment36. This approach may be useful to further enhance the specificity of the assay. ACKNOWLEDGEMENTS The authors acknowledge Dr. Victor Morozov for fruitful discussions. Funding from Council on grants of the President of the Russian Federation (grant 2697.2015.4 to Yu. M. Sh.) is also acknowledged. ASSOCIATED CONTENT: the sequences of oligonucleotides used; testing the immobilization efficiency and non-specific adhesion of magnetic beads; the description of the image processing procedure; force discrimination assay of partially and fully complementary DNA sequences; illustration of the specificity of the multiplex determination of three microorganisms. REFERENCES 1. Yang, S.; Rothman, R. E. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis. 2004, 4, 337–348. 2. Gavrilov, A. A.; Chetverina, H. V.; Chermnykh, E. S.; Razin, S. V., and Chetverin A. B. Quantitative analysis of genomic element interactions by molecular colony technique. Nucl. Acids Res. 2014, 42, e36.



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