1
Sequential DNA hybridisation assays by fast micromixing

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

2
The prospects of performing DNA hybridisation assays in a novel sequential scheme are explored in this article.

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

3
It is based on recording the kinetics of hybridisation on a microfluidic device measuring only 10 by 5 mm.

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

4
It contains a split channel system for fast mixing and a subsequent meandering channel to observe the evolution of the mixture by optical means.

Type: Object | Advantage: Yes | Novelty: New | ConceptID: Obj1

5
The problems of diffusion limitations in the laminar flow regime are overcome by reducing the average diffusion distance to a few micrometers only.

Type: Object | Advantage: No | Novelty: New | ConceptID: Obj1

6
DNA oligomers (20-mers) of different sequences were injected on the chip for mixing.

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

7
The detection of hybridisation was based on the fluorescence of DNA-intercalating dyes.

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

8
Two modes of operation were investigated.

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

9
First, the samples were injected into the micromixing device at a high flow rate of 40 µl min−1.

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

10
When the sample passed through the actual micromixing unit, the flow rate was reduced to allow for measurement of fluorescence levels at various steady-state reaction times in the range of 2–15 s, as defined by the channel geometry.

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

11
Using this continuous flow approach, photobleaching of fluorophores could be avoided.

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

12
In a buffer containing 0.2 M NaCl, 2 base-pair mismatches could routinely be detected within 5–20 s.

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

13
Single base-pair mismatches were successfully identified under low salt conditions.

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

14
In the second mode, the flow was completely stopped and the evolution of the total fluorescence signal influenced by the hybridisation of oligomers and photobleaching was observed.

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

15
Whereas the sequence-dependent effects remained unchanged, the assay times between the mixing of two oligomers and clear identification of their hybridisation properties could be reduced down to a maximum of 5–7 s, in some cases even below 1 s.

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

Introduction

16
DNA hybridisation reactions are at the core of biological and medical analysis.

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

17
Performed in vast numbers, one of the most valuable platforms are DNA microarrays where probe oligomer sequences representing genes of interest are immobilised and incubated with a mixture of unknown target DNA.

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

18
Fluorescent labelling techniques are predominantly used to identify the sites where probe and target sequences match.

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

19
The performance of DNA microarrays is impressive as integration with microsystem technology progresses to increase the number of probe oligomer per unit area.1,2

Type: Method | Advantage: Yes | Novelty: Old | ConceptID: Met2

20
Thereby, the information density is increased considerably and enables large-scale genomic analysis such as gene expression profiling of whole organisms.3–5

Type: Method | Advantage: Yes | Novelty: Old | ConceptID: Met2

21
Although it seems that many application needs in industrial research and high-throughput screening can be satisfied with microarrays, there is an on-going interest in alternative DNA hybridisation technology, e.g. hybridisation-based sensors that depend on other sensing devices like fiber optics,6 surface plasmon resonance sensors7 or acoustic wave sensors.8

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

22
Especially attractive solutions were presented particle-based probe oligomer immobilisation schemes.9,10

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

23
Yamashita et al. proposed a microfluidic system based totally on laminar flow, where the DNA hybridisation occurs at the interface between the streams.

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

24
By tailoring the geometry of channel bends on their chip, only the newly formed and fluorescence-labelled DNA duplexes migrate into the bulk region of the second laminar stream for detection.11

Type: Method | Advantage: Yes | Novelty: Old | ConceptID: Met5

25
Another interesting contribution from Tamiya's group suggested the use of silicon micromachining to create platelet-like particles for immobilisation whose shape characteristics alone could code for different functionalisation and still be easily identifiable in mixtures.12,13

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met6

26
However, there is a distinct lack of scale-up potential towards high-throughput applications in most of these approaches.

Type: Method | Advantage: No | Novelty: None | ConceptID: Met7

27
In some cases, the beads coated with probes could be sorted into a silicon frame and became individually addressable.14

Type: Method | Advantage: No | Novelty: None | ConceptID: Met7

28
We wish to present a different approach with potential for high-throughput application by combining purely solution-based DNA hybridisation reactions with the use of a microfluidic system.15,16

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

29
This device overcomes the mixing problem that is inherent to most microfluidic systems which work in the laminar flow regime and is based on the fine distribution of two laminar inlet flows (Fig. 1).

Type: Method | Advantage: Yes | Novelty: New | ConceptID: Met1

30
DNA hybridisation assays are performed in series by injecting the sample solutions one after the other and mix them rapidly on the chip.

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

31
Single nucleotide polymorphisms (SNP) are detectable.

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

32
If such a scheme is to be used for a large number of analyses in sequence, the single assay has to be performed within a short time to become comparable to the enormous multiplexing performance of microarray technology.

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

33
The prospect of decreasing the time of one assay to the order of 1 s is presented.

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

34
Potentially, this technology could allow for many thousand assays within just a few hours.

Type: Method | Advantage: Yes | Novelty: New | ConceptID: Met1

35
By using solution-based DNA chemistry,17,18 the need for immobilisation schemes is eliminated.

Type: Method | Advantage: Yes | Novelty: New | ConceptID: Met1

36
Due to restrictions of molecular diffusion near surfaces, the kinetics of heterogeneous hybridisation assays are slower, as characterised in literature.19–22

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

37
The detection of DNA hybridisation is based on the fluorescent dye PicoGreen that selectively enhances its fluorescence upon binding to double-stranded DNA (dsDNA).

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

38
Typically, such intercalating reagents are used for DNA quantification23–27.

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

Experimental procedures

Instrument setup

39
The setup consisted of a Leica DM IL inverted microscope with fluorescence detection equipment as depicted in Fig. 1c.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met8

40
The glass-silicon-glass sandwiched micromixing device was secured in a custom-made aluminium holder by pressing PEEK capillaries and silicone ferrules (Upchurch Scientific, Eugene, Oregon, USA) into the access holes of the microchip.

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

41
There were two light sources installed, one a halogen lamp for bright-field imaging, the other lamp a high-performance Hg-lamp (50 W) for fluorescence excitation.

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

42
Various neutral density filters could be inserted before the fluorescence filter set in order to attenuate the excitation light.

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

43
Connected to the microscope was a photomultiplier tube (PMT) Cairn Integra (Cairn Research Ltd., Faversham, UK).

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met9

44
Its preamplifier analog voltage output was connected to a National Instruments PCI DAQ card for direct digitalization of the PMT data.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met9

45
Liquid pumping was done using a Harvard PhD 2000 syringe pump equipped to hold up to two Hamilton syringes.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met9

46
One input line was connected to a Rheodyne EV750-100 6-port injection valve (Rheodyne LLC, Rohnert Park, California, USA) which was equipped with a 20 µl sample loop.

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

47
A video camera for capturing live and still images was installed on the top port of the PMT mirror assembly.

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

48
By the use of dichroic mirrors which split the light at 600 nm, the camera remained connected in parallel to the PMT so that detection window and positions could be checked shortly before and during measurement.

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

49
The detection window was defined by an adjustable rectangular aperture of 160 by 200 µm.

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

50
The fluorescence light was collected at highest magnification through a 32× objective (NA = 0.4).

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

51
The whole setup was computer-controlled using LabView and was placed in a cloth-clad cubicle in order to avoid stray light.

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

Micromixer flow visualisation

52
A solution of 0.385 mM fluorescein isothiocyanate (FITC) dissolved in MeOH was used in a mixing experiment with water.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

53
The components were mixed at a total flow rate of 120 µl min−1.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

DNA hybridisation assays

54
Gel-filtered DNA oligomers were synthesized by Eurogentech, Romsey, UK and used as received.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

55
The lyophilised DNA oligomers were reconstituted using sterile and RNAse free water.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

56
The following sequences were used for the present work, a base sequence A: 5′-GTTTATTAATGCGGCCCGCG-3′, its perfect match A′: 5′-CGCGGGCCGCATTAATAAAC-3′, as well as sequences containing increasing numbers of base pair mismatches, A-1: 5′-CGCGGGCTGCATTAATAAAC-3′, A-2: 5′-CGCGAGCCGCATTGATAAAC-3′ and A-5: 5′-CGTGAGACGCACTGATATAC-3′.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

57
Location of mismatches are indicated in bold-oblique letters.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

58
The hybridisation properties of various oligomer mixtures were also confirmed by DNA sizing on an Agilent 2100 bioanalyser.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

59
To ensure temperature stability and constant lighting conditions etc., each experiment consisted of different injections done in the same run within a few hours.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

60
PicoGreen was purchased from Molecular Probes Inc., Leiden, Netherlands.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

61
For a typical experiment, 3 ml of buffer solution containing 34.5 mg NaCl (0.2 M) and 8.9 mM tris-borate/0.2 mM EDTA at pH 8.3 were prepared and put under vacuum for degassing.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

62
To this buffer, the appropriate amount of oligomer and PicoGreen dye (typically 5 µl of PicoGreen diluted in water 1:10) was added shortly before the experiment.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

63
The first syringe connected directly to one inlet usually contained 450 µl of a 0.8–8 µM (5–50 µg ml−1) target oligomer solution, the syringe supplying the other inlet contained 450 µl pure buffer and was used as carrier stream through the Rheodyne injection valve.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

64
Probe oligomer solutions for injection were at the same concentration unless indicated otherwise.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

65
Between experiments, the chip was filled with liquid in order to avoid air bubbles forming and occasionally flushed with buffer and EtOH/MeOH or 2-propanol to keep the microchannels clean.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

66
Calibration experiments to assess the detection limit of dsDNA within a large background of ssDNA were performed.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

67
All solutions were prepared with excess oligomer to mimic the initial stages of hybridisation with the corresponding amount of unreacted oligomer.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

68
A 5 µM stock solution of dsDNA (A:A′) was diluted to 1, 2, 20, 40, 80, 160 × 10−8 M concentrations to which another equivalent of A was added to bring the total oligomer concentration in the system to a total of 8 µM (comprising dsDNA and ssDNA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

69
This time, the solutions were injected on the microchannels through the outlet since the micromixing function was not necessary.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

70
Fluorescence levels were recorded for both stopped flow and continuous 40 µl min−1.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

71
As the results of the calibration run indicate, dsDNA concentrations of 200–400 nM could readily be detected (not shown).

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

72
Where applicable in terms of comparable experimental conditions, a linear fit to this calibration was used to convert the PMT voltage scale to concentration units.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met10

73
The PMT data was first normalised to the measured basic fluorescence level of one oligomer/PicoGreen solution mixed 1 : 1 with buffer.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met10

Results and discussion

74
The high efficiency of mixing in the laminar flow regime is brought about by splitting each of the two inlet streams into 16 smaller substreams which then are joined and recombined (Fig. 1a).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

75
Details concerning the design and microfabrication procedures of the device are given elsewhere.28

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

76
Briefly, channels and through-holes were micromachined into a silicon wafer from both sides by deep reactive ion etching (DRIE) using three photolithography masks.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

77
The silicon wafer then was sandwiched by anodic bonding of a plain glass wafer at the bottom and a glass wafer featuring sand-blasted access holes on top and finally diced to yield single chips.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

78
The channel surfaces had not been functionalised in any way.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

79
Hence, a layer of adsorbed oligomers on the open SiO2 surfaces had to be expected.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp1

80
Due to the excitation light source containing significant intensity in the UV range, an irreversibly bound monomolecular layer is likely to have formed.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp1

81
However, due to the dynamic operation mode featuring only short interruptions of a continuous flow, the immensely larger detection volume, the DNA concentrations used and frequent flushings, no evidence of adverse effects on the fluorescence signals was found.

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

82
In addition, the intense excitation light source was constantly illuminating the channel system (see positioning of aperture in Fig. 1c), thus quenching any remaining fluorescence from possible adsorbates by photobleaching.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met11

83
With the present system geometry, Reynold numbers of Re = 1.6 to 4.8 have been calculated for the flow rates of 40 to 120 µl min−1 of aqueous oligomer solutions used within this project.

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

84
In the absence of turbulence at Re < 2,000, the only transport mechanism for mixing is diffusion.

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

85
The splitting of flows therefore reduces the average diffusion distance to approx.

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

86
10 µm, see also Fig. 1b where micrographs of the stratified laminar flow pattern after mixing fluorescein isothiocyanate (FITC) with water are shown.

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

87
A meandering channel allows for downstream observation of the flow, e.g. after 30 ms the laminar pattern has been washed out by diffusion.

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

88
The splitting of flows leads to a significant decrease in mixing times down to the order of ms, since the diffusion distance x follows a root law as function of diffusion coefficient D and time t

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

89
A literature overview of some diffusion properties in water for various molecules of interest are summarised in Table 1.

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

90
Smaller molecules like FITC easily cover 7–8 µm within 100 ms, whereas DNA oligomers and double-stranded pieces of 20 bp length take up to approximately 1 s for the same distance.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

91
This is due to their diffusion coefficient being approx.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

92
10× smaller.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

93
It is important to note that these values may vary depending on the actual experimental conditions like buffers, pH, temperature, DNA sequence and functionalisation etc.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

94
Therefore, these literature values were used as a guideline only.

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

95
All the characteristics addressed above led to the decision to vary the flow rate in the present work by mixing probe and target oligomer quickly using a high flow rate and subsequently slowing down or stopping the flow to allow for enough time to observe the evolution of the DNA hybridisation reaction.

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

96
The downstream channel volume of 480 nl would only allow for a time window of 600 ms at typical flow rate of 40 µl min−1.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

97
Therefore, these reductions of the flow-rate or stop-flow schemes were required.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met12

98
Since the detection of hybridisation is based on intercalating dyes which increase the quantum yield of fluorescence upon interacting with dsDNA, the following contributions to the overall fluorescence level can be expected: (i) the fluorescence of the intercalating dye in solution without DNA interaction, (ii) interaction of the dye with ssDNA, (iii) intercalation into oligomer-dimers, (iv) intercalation into dsDNA.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp2

99
Of these contributions, (i) should be negligible29 (ii) is expected to remain constant throughout the experiments, or, in the case of matching oligomers, is expected to become negligible as ssDNA is consumed by hydridisation, (iv) desirably the strongest and (iii) will have to be controlled carefully, since it may vary depending on the probe sequences of the oligomers in use.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp2

100
Newer intercalating dyes like PicoGreen seem to fulfil these requirements, especially since the brightness increase upon binding to dsDNA is reported to be 2 orders of magnitude.

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

Continuous-flow experiments, detecting SNPs

101
A typical experiment mixing oligomer A with A′ is depicted in Fig. 2a.

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

102
At 40 µl min−1 the injection valve is switched to its inject position for 10 s, injecting approximately 3.3 µl of probe oligomer solution.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

103
Concurrently with switching the valve back to the load state, the flow is abruptly reduced to a flow rate of 2 µl min−1.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

104
After 10–20 s another steady state of the fluorescence signal is reached.

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

105
For demonstration of the repeatability of this flow rate change, the flow rate is temporarily brought back to 40 µl min−1 for just a short moment (at 45 s).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

106
The signal recovers to the same steady-state level after that.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs9

107
The experiment is ended at 80 s by flushing the system at 40 µl min−1.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

108
Both oligomer concentrations were 2.4 µM.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

109
The observed difference in fluorescence intensity before and after the flow rate reduction step, ΔPMT, was evaluated to detect oligomers that match or, respectively, mismatch in varying degrees.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met13

110
The ΔPMT values of sequence-dependent experiments are shown in Fig. 2b.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

111
The same experiment as described above was performed with various mismatch combinations.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met14

112
Matching oligomers A/A′ and 1-mismatch A/A-1 could not be distinctly resolved.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs11

113
This is attributed to the high NaCl concentration of 0.2 M which was initially chosen to maximise the reaction rate at fixed oligomer concentrations.

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

114
The error of multiple injections was dominated by the fluctuations of the mercury-arc lamp that was used.

Type: Method | Advantage: No | Novelty: New | ConceptID: Met14

115
However, if the NaCl content is reduced to 40 mM, the stringency of hybridisation is increased.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

116
Using these conditions, single base-pair mismatches A/A-1 were successfully distinguished from A/A′ at 4.8 µM oligomer concentrations (Fig. 2c).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs12

117
As both syringes were driven by the same pump, the mixing ratio was fixed to 1:1.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met15

118
However, due to individual pressure variations upon flow rate change, deviations from this 1:1 ratio had to be taken into account.

Type: Method | Advantage: No | Novelty: New | ConceptID: Met15

119
Therefore, the initial response always carries some unwanted spike which was characterised by observing the effect of flow-rate change without injecting a sample.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs13

120
In those cases, the corresponding fluorescence response of only the change in flow rate without sample injection is also displayed.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs13

121
The difference between the two steady-state levels is indicative of the hybridisation reaction rate.

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

122
As Fig. 2c also shows, the higher concentrations used in the single base-pair mismatch experiments yields a much shorter settling time of only 5 s to reach flow equilibrium.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs14

Stop-flow experiments

123
A sharp increase in fluorescence brightness was obtained when injecting the complementary 20-mer A′ to the basic sequence A and stopping the flow.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs15

124
The injection time of 10 s was again chosen in order to fill the channel downstream of the mixing unit long enough to perform several stop-flow measurements.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp10

125
A series of three injections is shown in Fig. 3a for [A] = constant = 8 × 10−6 M and [A′] = 8 × 10−6 M, resp.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs15

126
4 × 10−6 M and 1.6 × 10−6 M. The curves each show the behaviour after stopping the flow 3 times and give an impression of the reproducibility.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs15

127
From top to bottom, the dilution of [A′] is shown.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs15

128
Clearly, the steady-state fluorescence level follows the dilution series quantitatively.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs15

129
The hybridisation kinetics of DNA oligomers can be described as a reaction following second order kinetics with a rate constant k1 of approximately 5.7 × 105 M−1s−1.18,30

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

130
The time-law of dsDNA formation for equal oligomer concentrations is given by where c0 is the initial concentration of both oligomers and k1 the rate constant.

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

131
Calculation of the 90% conversion value yields a value of t90 = 1.98 s.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

132
Therefore, considering the specific conditions used in the experiments, the hybridisation should be faster than the mixing process by diffusion.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp3

133
One expects to measure the response of the mixing system rather than the hybridisation kinetics themselves.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp3

134
As Fig. 3a confirms, the times after the stopping of flow required to reach a steady fluorescence level do not change by diluting the concentration of one oligomer.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs16

135
Linearisation to the root law of diffusion (1) of the time-dependent response shows that the hybridisation is indeed in the diffusion-limited regime for 20 s after which the increase in fluorescence deviates from the root law (Fig. 3b).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

136
Possible explanations for this deviation are the completion of the mixing process or the onset of photobleaching as the intense excitation light is constantly focussed on the same volume element of the liquid after halting the flow.

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

137
The t90 values scale linearly with concentrations.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs17

138
Hence, it has to be expected that using lower concentrations will eventually lead to a kinetic limitation of the response.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp4

139
If this micromixing/stopped flow scheme is going to be used for genomic analysis, the dependence of the response to variations in sequence will be crucial.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot3

140
Examples of sequence series are given in Fig. 4.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs18

141
The A sequence was provided to the first inlet and the other oligomers were injected via the Rheodyne valve to the second inlet with injection times of 10 s.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp11

142
A′ and A-1 containing one mismatched base pair coincide, whereas the first differences become evident for the two-mismatch oligomer, A-2.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs19

143
In the case of A-5, the fluorescence increase is distinctly lower reflecting the thermodynamic equilibrium being shifted to the oligomer side.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res9

144
True single-nucleotide-polymorphism (SNP) detection could again not be achieved due to the NaCl content (0.2 M).

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

145
However, the same way as control probe sequences containing an additional mismatch close to the target mismatch are included in microarray applications, such an approach could equally be applied for the proposed micromixing approach.

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

146
Further variations in buffer composition also lead to less ‘mismatch-tolerant’ and more stringent hybridisation conditions.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs20

147
High-throughput application of this scheme demands for a quick succession of assays, e.g. another assay every 1–5 s.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot4

148
It is not necessary to wait for the hybridisation to complete for obtaining the information whether two particular probe to target sequences match.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

149
This will constitute an optimisation problem between fast detection and the resulting sequence-related levels of confidence.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

150
Parameters to consider are the diffusion-controlled mixing or the kinetics of hybridisation and the sensitivity of detection, especially the detection of small dsDNA amounts in a large background of oligomers.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot4

151
A true high-speed approach is shown in Fig. 5, where multiple stops on A mixed with A′, and A mixed with A-5 are depicted.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs21

152
Particular to this experiment was the removal of all ND filters to have the highest excitation light intensity that the setup permitted.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met16

153
Photobleaching effects now become very significant and the hybridisation/intercalation process produces just enough additional fluorescence to temporarily outweigh the photobleaching.29,31

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs21

154
Therefore, the signal obtained only increases in the case of matching oligomer sequences.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs21

155
In the case of mismatching sequences, A-5 to A, only a decay of fluorescence is recorded.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs22

156
First, such a scheme could provide for a signal with less ambiguity than the previous ones shown before by exhibiting a clear increase/decrease of fluorescence brightness.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con6

157
Second, the magnified extracts that show the onset of the signal upon halting the flow are only 1 s, clearly indicating the possibility to discriminate matching/nonmatching situations with a signal change in the range of 10% within the first second of observation.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con6

158
We have demonstrated an alternative approach for performing DNA hybridisation assays in sequence.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met17

159
The enabling tool is a microsystem of passive mixing channels with subsequent observation channel.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met17

160
DNA hybridisation at high concentrations was found to be diffusion-limited.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs23

161
Sequence dependent signals down to SNP-level were successfully recorded.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res10

162
A mode of operation suitable for high-throughput applications was found, where the signal shows within 1 s whether two oligomers match.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res10

163
Whereas technological difficulties such as the periphery providing a steady stream of samples still need to be addressed, we believe this method could provide for a very flexible and versatile platform for genomic analysis.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

Acknowledgements

164
M. Heule thanks for funding, by a postdoctoral fellowship award, the Swiss National Science Foundation (SNF).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

165
Upchurch Scientific is acknowledged for providing a reference chip holder.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10