Sequence-selective DNA detection using multiple laminar streams: A novel microfluidic analysis method

Abstract

On-site detection methods for DNA have been demanded in the pathophysiology field. Such analysis requires a simple and accurate method, rather than high-throughput. This report describes a novel microfluidic analysis method and its application for simple sequence-selective DNA detection. The method uses a microchannel device with a serpentine structure. Sequence-specific binding of probe DNA can be detected at one side of the microchannel. This method is capable of sequence-specific detection of DNA with high accuracy. Single base mutations can also be analyzed. Combination of laminar stream and laminar secondary flow in the microchannel enable specific detection of probe-bound DNA.

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Introduction

Pathophysiological analysis requires a simple and accurate method, rather than high-throughput.¹ Current DNA analysis methods utilize an enzyme reaction^2,3 or DNA probe.^4,5 The latter technique is especially suitable for rapid DNA detection.¹ Several methods based on this technique have been developed using a groove binder⁶ or intercalator^1,7–9 as the hybridization indicator. However, these methods have not been applied generally to pathophysiology because of technical difficulty, limited applications, low accuracy, and high cost.^10,11

We have addressed microfluidic systems to develop a simple and accurate method for quick DNA analysis. Microfluidic systems offer superior controllability of fluidics.^12–14 The fluid forms a laminar stream at the straight part, whereas laminar secondary flow occurs at the turning point of a microchannel. This “laminar secondary flow” is the perpendicular flow to the principal laminar stream along the channel, and controlled easily by the channel structure and flow speed. Thus, we can create any kind of fluidic system by changing the channel structure.

Experimental

Microfluidic system

The acrylic microfluidic system chip (3 cm × 7 cm) used in this study was prepared by mechanical fabrication methods as reported in the previous paper.¹⁵ The microchannel on this chip has a cross-sectional plane whose size is 300 µm width and 200 µm depth, 2.0 mm diameter curve and 24 cm length (for Fig. 3A), or 1.5 mm diameter curve and 49 cm length (for Fig. 4A).

Chemicals

All probe and target oligonucleotides were obtained from Qiagen K. K. (Japan). The probe DNA was labeled by FITC at the 5′-end. Target DNAs 1–4 have a complementary sequence to probe DNA, 5 and 6 have a one-base mismatch mutation sequence, and 7 was prepared as a negative control. Sequences corresponding to probe DNA and target DNAs 1–6 were designed using part of a unique sequence of exon 4, on which the P72/R72 SNP resides, of cancer repression gene p53.¹⁶ Concentrations of these oligonucleotides were estimated from the molar absorptivities at 260 nm:^17–19 171 800 cm⁻¹ M⁻¹ for probe DNA, 98 300 cm⁻¹ M⁻¹ for target 1, 144 600 cm⁻¹ M⁻¹ for target 2, 186 000 cm⁻¹ M⁻¹ for target 3, 648 600 cm⁻¹ M⁻¹ for target 4, 101 800 cm⁻¹ M⁻¹ for target 5, 189 500 cm⁻¹ M⁻¹ for target 6 and 243 400 cm⁻¹ M⁻¹ for target 7.

Apparatus

A syringe pump (KDS230; Kd Scientific, MA) controlled all injections of solutions into the microchannel. Fluorescence intensity in sequence-selective DNA sensing was measured using fluorescence microscopy with a fluorometer (C7473; Hamamatsu Photonics K.K., Japan), Ar-gas laser (Stabilite 2017, Spectra-physics Inc., CA) and a longpass filter (OG515; Edmund Industrial Optics Co. Ltd., NJ).

Sequence-selective DNA detection

Probe and target DNA solutions were charged into the microchannel by syringe pumping simultaneously. Then fluorescence intensity of target solution side and probe solution sides were measured near the outlet of the straight part of the microchannel using the above described fluorescence microscopy. Detection was evaluated with a ΔF value.

Results and discussion

Fig. 1 outlines the analysis system. Two solutions, probe and target DNA solutions, were charged into the microchannel simply by syringe pumping. In a straight microchannel, mixing the two solutions simply depends on diffusion (ca. 1 µm s⁻¹). Therefore, double strands of target and probe DNA were formed around the interface area. The molecular weight increases by double strand formation; thereby the molecule localizes at the interface. At the curving part of the microchannel, internal force of the fluid produces laminar secondary flow within the channel. Such laminar secondary flow at the turn disrupts the interface and moves double-stranded DNA molecules to the outer side. When solutions were charged with double-stranded DNA, as shown in Fig. 1, we only have to compare the ΔF value, which is the background (B₀/A₀) subtracted ratio of fluorescence intensity at the probe DNA solution side (A) and target DNA solution side (B) of the microchannel. (A₀ and B₀ are the fluorescence intensities in the case of buffer solution instead of target DNA solution).

Fig. 1 Schematic principle and procedure of the sequence-specific DNA detection in the microfluidic system.

The microchannel design is very important for this analysis method. The microchannel design in this study has been optimized by advance examination.

Typical results were obtained using DNA molecules as shown in Fig. 2. First, we examined which position of the channel is suitable for detection (Fig. 3A, position a–e), using complementary sequence 3 and negative control 7. Fig. 3B shows that the complementary target gave higher intensity ratios than those of the negative control at positions c and e. On the other hand, such a difference was not observed at positions b and d. These results mean that specific detection of double-stranded DNA was enabled at the points after curves of even numbers. This can be explained by simulation of microfluidics. Laminar secondary flow disrupted the interface at the first curve, but the structural interface was restored by laminar secondary flow with opposite direction at the next curve. Thus, the difference could be observed at positions c and e, whereas no difference was obtained at positions b and d.

Fig. 2 Structures of probe and target DNAs 1–7 used in this study.

Fig. 3 (A) The microchannel structure used in this measurement and (B) detection of sequence-specific binding at any positions. ΔF values are shown as averages (n = 10). This microchannel has a 2 mm diameter curve and 24 cm length. The total flow speed is 40 µl min⁻¹ (1.1 cm s⁻¹). These measurements were carried out at 23 °C. Probe and target DNA solutions are 0.50 pM in 5 mM phosphate buffer (pH 7.0) and 50 mM NaCl.

Fig. 4 shows the results for target DNAs 1–7 at position f in two different temperature and solution conditions. At higher salt concentrations and lower temperature conditions (23 °C, 50 mM NaCl) such as Fig. 4B, ΔF values of targets 1–6, which are complementary and one base mismatch sequences are larger than those of target 7. In contrast, at lower salt concentration and higher temperature conditions (35 °C, 5 mM NaCl) such as in Fig. 4C, ΔF values of short (1) or one base mismatch (5, 6) sequences are relatively low compared to target 7. These results reflect the stability of the double strand in solution. We examined whether target molecule length affects analysis or not. We used 10mer to 70mer DNA as the targets (1, 2, 3, and 4). Fig. 4C indicates that ΔF value increased in proportion to the target molecule length at position f. These results demonstrated that the principle of our strategy can be applied for analysis. We also examined whether this method is applicable for single base mutation analysis or not. Experiments were performed using samples with one base mismatched sequences (5 and 6). Although the experiment was performed as in analysis of a complementary target, ΔF values were almost the same level as in the negative control 7 in Fig. 4C. Thus, we could detect single base mutations; our method is applicable for such SNP analysis. Accuracy of the analysis was also examined. The coefficient of validation for these analyses was nearly 5%. Usually, such a low coefficient of validation cannot be obtained by current technologies such as electrochemical methods utilizing immobilization procedures. In our case, simple syringe pumping might result in such high accuracy. The laminar stream in the microchannel arrives at steady state immediately. At such a steady state, there are no artifacts except for slight pulsation caused by syringe pumping. Therefore our method always offers high accuracy even at small ΔF values.

Fig. 4 (A) The microchannel structure used in this measurement and (B and C) detection of sequence-specific binding. ΔF values are shown as averages (n = 10). The error bars are standard deviations. This microchannel has a 1.5 mm diameter curve and 49 cm length. The total flow speed is 80 µl min⁻¹ (2.2 cm s⁻¹). These measurements were carried out at (B) 23 °C or (C) 35 °C. Probe and target DNA solutions are 0.50 pM in 5 mM phosphate buffer (pH 7.0) and (B) 50 mM or (C) 5 mM NaCl.

Conclusion

We have developed a novel analysis method of sequence-selective DNA detection using microfluidics. This method does not utilize immobilization procedure, but simply uses syringe pumping. By merely injecting target and probe DNA solution, we were able to detect sequence-specific binding of probe and target DNA. The simple operation enables highly accurate DNA analysis: even a single base mismatch could be detected. These features might be suitable for further application of our method in biomedical fields.

Acknowledgements

This work was supported by grants from the MEXT of Japan.

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