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Back | Show only these items: | Sequence-selective DNA detection using multiple laminar streams: A novel microfluidic analysis method

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

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

On-site detection methods for DNA have been demanded in the pathophysiology field.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

Such analysis requires a simple and accurate method, rather than high-throughput.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

This report describes a novel microfluidic analysis method and its application for simple sequence-selective DNA detection.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

The method uses a microchannel device with a serpentine structure.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

Sequence-specific binding of probe DNA can be detected at one side of the microchannel.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

This method is capable of sequence-specific detection of DNA with high accuracy.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

Single base mutations can also be analyzed.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

Combination of laminar stream and laminar secondary flow in the microchannel enable specific detection of probe-bound DNA.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

Introduction

Pathophysiological analysis requires a simple and accurate method, rather than high-throughput.¹

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

Current DNA analysis methods utilize an enzyme reaction^2,3 or DNA probe.^4,5

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

The latter technique is especially suitable for rapid DNA detection.¹

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

Several methods based on this technique have been developed using a groove binder⁶ or intercalator^1,7–9 as the hybridization indicator.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

However, these methods have not been applied generally to pathophysiology because of technical difficulty, limited applications, low accuracy, and high cost.^10,11

Type: Method | Advantage: No | Novelty: Old | ConceptID: Met2

We have addressed microfluidic systems to develop a simple and accurate method for quick DNA analysis.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

Microfluidic systems offer superior controllability of fluidics.^12–14

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

The fluid forms a laminar stream at the straight part, whereas laminar secondary flow occurs at the turning point of a microchannel.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

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.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

Thus, we can create any kind of fluidic system by changing the channel structure.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

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

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met3

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).

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod4

Chemicals

All probe and target oligonucleotides were obtained from Qiagen K. K. (Japan).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

The probe DNA was labeled by FITC at the 5′-end.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

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.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod5

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 p.53¹⁶

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met4

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

Apparatus

A syringe pump (KDS230; Kd Scientific, MA) controlled all injections of solutions into the microchannel.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

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).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

Sequence-selective DNA detection

Probe and target DNA solutions were charged into the microchannel by syringe pumping simultaneously.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

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.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Detection was evaluated with a ΔF value.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Results and discussion

Fig. 1 outlines the analysis system.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod6

Two solutions, probe and target DNA solutions, were charged into the microchannel simply by syringe pumping.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

In a straight microchannel, mixing the two solutions simply depends on diffusion (ca. 1 µm s^–1).

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod7

Therefore, double strands of target and probe DNA were formed around the interface area.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod8

The molecular weight increases by double strand formation; thereby the molecule localizes at the interface.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod8

At the curving part of the microchannel, internal force of the fluid produces laminar secondary flow within the channel.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod6

Such laminar secondary flow at the turn disrupts the interface and moves double-stranded DNA molecules to the outer side.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod6

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.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod9

(A₀ and B₀ are the fluorescence intensities in the case of buffer solution instead of target DNA solution).

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod9

The microchannel design is very important for this analysis method.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

The microchannel design in this study has been optimized by advance examination.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

Typical results were obtained using DNA molecules as shown in Fig. 2.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

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.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Fig. 3B shows that the complementary target gave higher intensity ratios than those of the negative control at positions c and e.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

On the other hand, such a difference was not observed at positions b and d.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

These results mean that specific detection of double-stranded DNA was enabled at the points after curves of even numbers.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

This can be explained by simulation of microfluidics.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod10

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.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod10

Thus, the difference could be observed at positions c and e, whereas no difference was obtained at positions b and d.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod10

Fig. 4 shows the results for target DNAs 1–7 at position f in two different temperature and solution conditions.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

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.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

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.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

These results reflect the stability of the double strand in solution.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

We examined whether target molecule length affects analysis or not.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa2

We used 10mer to 70mer DNA as the targets (1, 2, 3, and 4).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Fig. 4C indicates that ΔF value increased in proportion to the target molecule length at position f.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs7

These results demonstrated that the principle of our strategy can be applied for analysis.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

We also examined whether this method is applicable for single base mutation analysis or not.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa3

Experiments were performed using samples with one base mismatched sequences (5 and 6).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

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.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

Thus, we could detect single base mutations; our method is applicable for such SNP analysis.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

Accuracy of the analysis was also examined.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa4

The coefficient of validation for these analyses was nearly 5%.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

Usually, such a low coefficient of validation cannot be obtained by current technologies such as electrochemical methods utilizing immobilization procedures.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

In our case, simple syringe pumping might result in such high accuracy.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

The laminar stream in the microchannel arrives at steady state immediately.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

At such a steady state, there are no artifacts except for slight pulsation caused by syringe pumping.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

Therefore our method always offers high accuracy even at small ΔF values.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

Conclusion

We have developed a novel analysis method of sequence-selective DNA detection using microfluidics.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

This method does not utilize immobilization procedure, but simply uses syringe pumping.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met5

By merely injecting target and probe DNA solution, we were able to detect sequence-specific binding of probe and target DNA.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

The simple operation enables highly accurate DNA analysis: even a single base mismatch could be detected.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

These features might be suitable for further application of our method in biomedical fields.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8