Flows in micro fluidic networks: from theory to simulation

Danny vam Noort Ecology and Evolutionary Biology Dept., Guyot Hall, Princeton University. Princeton. 08544 NJ, USA dnnnvriiinrinceton.2dii John S. McCaskill Fraunhofer Society, BioMIP, lnstitutszentrum Birlinghoven, SchloP Birlinghoven, 53754 Sankt Augustin. Germany inccnskill[~biomin.~~.de

Abstract- When complex flow structures are designed, such as in DNA computing [l],it is essential to be able to predict the flow pattern of the solutions in the fluidic network. A model based on the resistance of the channels and flow velocities of the inlets can eliminated re-iterative design steps. We have constructed a symbolic model (using Mathematicam)to determine the desired flow pattern based on the equations of Ohm and Kirchoff. The values from this simulation were used in a flow simulation program.

were made with a microfluidic simulation software package from Coventor'" (Coventor Inc.. NC. USA).' First we present the necessary equations to calculate the resistance of a rectangular shaped tube after which the reactor modules and their working principle will he introduced.

2 Theory
When flows are laminar. the non-linear term v.R in the Navier-Stokes equation becomes zero (e.g. Happel and Brenner 161). Exact solutions of the remaining linear equations are then possible, especially if the flow is steady as is the case considered here. Indeed. the microfluidic architecture was deliberately chosen to avoid dynamic flow switching. For flow in the x-direction. the Navier-Stokes law implies:
V Z U = -1 dp

1 Introduction
Complex flow structures. like the DNA-computer we are developing [l.Z]. are difficult to construct without the use of calculation and simulation tools. Simple open networks. i.e.. with one input and one output can be constructed ad hoc. However. when there are multiple inputs and outputs. it is a necessity to know the exact ratio between the flows and volume. The fluidic network should he balanced by determining the channel resistance and the input velocities required to realize the direction and volume of flows. The resistance and flow properties are calculated in a similar fashion to that used in electric circuit theory, i.e. the same laws apply. The resistance can he calculated using Ohm's law while the first law of Kirchhof states that the sum of currents is zero in nodes of the circuit. The analogy between fluidic and electric circuits is that the electric resistance (depending on the wire dimensions. the dielectric properties of the material) corresponds to the hydrodynamic flow resistance (depending on the channel dimensions, the material properties of the channel) while the current (the flow rate of electrons in the wire) corresponds to the flow velocity (the flow rate of the liquid in the channels). The voltage drop over a wire corresponds to the pressure drop over a channel. The use of equivalent resistance circuits in microfluidic design was pioneered in the diploma thesis of Arne Bochman [3] (Univ. Jena. 1996). Other recent research use theoretical models to perform calculation, e.g., flow rates of a membrane pump or the impedance for microfluidic systems, were performed by Bardell and Forster [4,5]. In our model the resistances of the micro channels were calculated algebraically using the computer propram Mathematicam (Wolfram Research Inc, IL. USA). assuming an ideal situation. Visualisation of flows

(1)

P dx

with U is the flowrate a n d p is the viscosity. The pressure gradient 4 is constant for a steady state
dx

and is written as

*=-P dx I

(2)

where ~p > 0 is the pressure difference between the ends of the channel of length 1. The end effects are neglected. So combining (1) and (2) gives v f u = -I AP (3)
P '

The volumetric flow rate Q is: i AP Q=

R I

(4)

where R is the specific resistance. The total resistance over the channel is
R, = RI

(5)

where I is the length of the channel. Substituting this in eq. (4) gives -dp=R,Q (6) (which corresponds to Ohm's law Al'=RI ). In the case of a rectangular channel with sides a and h. the volumetric flow rate is [SI:

0-7803-7804-0 1031117 00 D ZOO3 IEEE

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o=*lub - pi 24

__ 4 8

;1

(Q 2 + b 21(7)

n5n=1(2n-ly

to determine the resistances and the desired inputs and output velocities. To prevent the beads from flowing to other STMs. a bead-barrier has been added to the design, which stops the beads from disappearing down the channels but creates additional hydrodynamic resistance.
A

[n"/un~(~~:+b'/anh(~;o:l

By substituting eq 7 in eq 6, the resistance of the channels can be calculated.

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I /
j
1

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3 Selection procedure
In DNA Computing [7]. DNA sequences are used as information carriers and as tools for the calculations. In the standard approach. each DNA sequence encodes binary sequence ( S ) . Different DNA sub-sequences are selectors used to represent single bit values (0. 1) at a certain position in a sequence (S,, i=1.2,3 ...). After each selection step. the sub-population is passed on to the next selection step. Each selection module is coded with short single stranded selection-DNA strand (ssDNA) from a finite set of predefined sequences. A selection is made by picking out that word which has the desired bit necessary for further processing.

Wa

out
N

Ti"

dH

4 Microreactor Structure
The selection procedure described above can be implemented in a microreactor. To this end we have developed a selection transfer module (STM) which is able to make positive selections from a population of specific DNA sequences. The principle of the selection module is shown in Fig. 1. To transfer actively the selected DNA sequences to the appropriate output. they are conveyed from one flow to another by moving paramagnetic beads, on which single stranded selectorDNAs (reverse complementary to a sub-sequence) are immobilised [I]. The DNA strands in the template solution hybridise to the selector-strands and are thus transferred to another channel in the microflow reactor where they are denatured and passed on to the next selection module (Fig. la). To optimise the transfer of only the appropriate sequences. a washing step has to be performed so as to rinse off the non-specifically bound DNA sequences. Denaturation is performed by using an alkali solution (NaOH), adjusted in concentration to the common melting temperature of the hybridised DNA strands. Because of the change in pH at the denaturation step, a subsequent neutralisation step is necessary after each selection stage before flowing the selected DNA into the next module. Here the wash is performed with the neutralisation solution. This is only possible if the flows have the correct flow pattern to do an adequate mixing (Fig. lb), which depends on the channel resistance in the system. Therefore a microreactor model has to be made

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5 Microreactor Model
The characteristics of the microfluidic system are dependent on the particular class of problems being addressed. In this case. u'e have designed a DNA computer to solve a particular NP-complete family of problems: the maximum clique graph problem. Details for this procedure can be found in McCaskill [ I ] and van Noort er. a/ [ 2 ] . The selection transfer module to be designed was shown in Fig. I .

Since we are working under different buffer conditions (denaturation solution should be neutralised to allow hybridisation conditions again in the next microreactor), ratios between the different buffers should be fixed and have been chosen l : l . This means that the velocity v, and v3, should be equal. Other constraints are that the pressure p7 is zero (there is the assumption that there is no residual pressure on the output relative atmospheric). The calculation is being made with R34 as a variable and all the other values fixed. If all the other resistors are set to 1 (these values being arbitrary. since we are only interested in the ratio between the resistors and velocities) except Rsh=3 and the velocity v2=4vI. then R,=4 and v,,=2vl. Using the values of these parameters gives us the correct fluidic network for the desired flow behaviour. Table I shows all the resistors and flow velocity values.

6 Flow Simulation
To visualise the calculated resistances and velocities used to design the microflow system, flow simulations were made with CoventorTM. As mentioned before. the values of resistance were taken as ratios and not as absolute values. Supply channels are needed to deliver the solutions to the STMs. To ensure that the resistance of the supply and waste channels don't corrupt the overall resistance. they should be at least 3 times wider with the same depth main channels.

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I,

A sche,nnric rqwrsriirarion of rltc firicroflm ~yncror cmsisriq qf 7 r.esismrs. Ourpur 7 is con,irc~rerlru irzprir 3 iri order- io ollou. colculnrions /o l ,for.n seyrrence qf sac11 w
ImClOlJ.

Fiyre 2

Figure 2 shows the module represented by resistors used for the calculation of the channel resistances and flow velocities. There are 3 inputs (template. neutralisation. de-hybridisation) and 2 outlets (waste and template). The horizontal part is the reaction chamber in which the selected DNA strands are transferred by magnetic beads from left to right where they are released to the template output. The network has been made iterative just for the calculations. which means that template out is connected to template in (node 4 is connected to node 3). In reality the problem is limited to a finite number of these reactors in sequence. To solve the problem we are working on. we need at least 15 of these reactors in sequence.
Tablr I

The cliannel resistances nnd flow i , ~ l o c i r i ~ i

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cnsc w h o r rirr

cliafiiiels wirlr the ,sum widlli as the rnnirz rlmnnels: supply c/iaiiriels are 3 rimes wider,

B slior~s lhe

This number is based on simulations concerning the distribution over side channels with fixed width as function of the width of the supply channel (Fig. 3). Figure 4 shows the flow velocity for the given side channels as a function of the supply channel thickness. Where the points overlap, there is a uniform flow distribution. The insert gives the distance of the fired side channels from the top of the network. as shown in Fig. 3.
+a +b c
16 ',

part of the STM. The curve C shows the ratios of these velocities. Because the flow in the reactor (vW)i s 3 times that of the input I (vI). the ratio between these velocities in the ideal case should be 3 when the resistance value is one. When the resistance in the reactor changes. the mixing ratio will alter. However. this ratio can be compensated with the inputflow velocities v,. v:. v3 (data not shown). This gives a certain amount of flexibility in the production stringency.

7 DNA computer
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In general the design of the DNA-computer is determined by the problem type and algorithm choice. which is also reflected in the number of reactors. For short term convenience, the number of STMs can be matched to the problem studied, but this is not a principle limitation. In order to allow the magnetic beads to move uniformly from left to right in all the STMs over the entire microflow reactor, alternate STMs are mirrored. When the DNA-sequences denatures from the beads. the beads in the next module will be in the correct place for hybridisation to take place. To solve a 6-node clique instance as described in [2]. 15 modules are needed. A 16"' module has been added. as a dummy, to balance the resistance of the 1 5 ' ~ module. which was found necessary when analysing the simulation data. Figure 6 shows the complete architecture of the DNA computer. The round pads are bead injection pads, needed to insert into the reactor the paramagnetic beads functionalised with specific short selection DNA. The broad, vertical channels are supply channels which are etched on the back of the wafer. while the squares on these channels indicate the trough etching locations. The top layer of channels is a sequence of STMs as described previously.

8 Structure and Processing

The array of cascading STMS were fabricated using 8 multi-step wet etching procedure [SI [9] of 4" <I002 silicon substrates, which makes multiple structuring with different etching depths possible. Double sided lithography was performed using AR-P 5 I10 (Allresist GmbH, Germany). Anisotropic etching of silicon is performed in a 25% wt. TMAH (Tetramethylammoniumhydroxide, Riedel de Haehn) solution at 80C. The etched wafer is sealed at the back with an anodically bonded pyrex glass wafer. Capillary tubing (0.8 mm diameter) is attached through ultrasound drilled holes in the pyrex wafer and fixed with a specifically developed adhesion technique for polyethylene tubes. To deliver the beads tu the STMs, a PDMS layer is attached on the etched silicon. providing bead delivery pads and channels that run into the reactor chambers from the tup. After injecting the beads with a syringe. the pads are sealed. The front side of the microreactor array.

0.6

0.8

1.0

1.2

1.4

1.6

Resistance [a.".]

Figure 5 Tlrv ,olio fCi " rile j7oo. velocities qr IBi f rind r, ( A ) nr,function qfrltr resisiancp change iit Rr.

Simulations have shown that changes in the resistance of the channels affect the distribution of the solutions. Figure 5 shows the flow velocities in the reactor (A) and the input I (B)as function of the resistance in the reactor

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including the bead injection pads and the delivery channels. are made in PDMS. The following technological steps are performed. A multi-depth master was fabricated by photo-lithographic patterning of a negative 'photoresist (SU 8-50, Microlithography Chemical Corp. Newton. MA) using two different photoemulsion masks. A specified amount of PDMS prepolymer (Sylgard 184, Dow-Corning) is then cast against the master. Curing the polymer and releasing it from the master yields a replica containing the bead delivery stmctures required. The structures are sealed irreversibly by oxidising the PDMS mould and the silicon wafer. after they are brought into contact.

9 Conclusions
Mathematical models and flow simulation tools provide a powerful base to design microfluidic networks. This allows a prediction of the flow patterns which is necessary to predict controlled mixing of reactants which is very important in biochemical processes. A suitable combination of analytical and simulation tools also saves time in the development process for the network.

Acknowledgments
The authors wish to acknowledge the support of the GMD and of the group's start-up grant (#01SF9952) from the German Ministry of Science (BMBF). We would like to thank Marlies Gohlke. and Patrick Wagler for their discussions and the manufacturing of the microfluidics network and Harald Mathis for providing the Rhodamine 6G and the laser set-up.

Bibliography
[ I ] McCaskill. J. S. (2001) Opricallj progrmitiririg DNA cor~rpritirrgirr rrricrqflou~ reucrors. Biosystems 59. 125-138. [Z] van Noort. D., Gast. F. -U. and McCaskill. J. S. (2002) DNA Coarputirrg in Microreactors. LNCS. in press. [3] Bochman. A. (1996). Diploma Thesis Univ. Jena. [4] Bardell. L. R.. Sharma. N. R.. Forster, F. K.. Afromowitr. M. A. and Penney. R. J. (1997) Desigrlirig high-perjonrrarlce nricro-prinrps based 011 r~ori-iiro~~ir~~ parrs. DSC-vol 621HTD-vol. 354. Microelectromechanical Systems (MEMS) ASME 1997. pp. 47-53. [ 5 ] Bardell. L. R. and Forster, F. K. (1998) Iri~pedurire.~ for desigrr of nricrofltridic syrenrs. Proc. of the Micro-TAS '98 Workshop. Banff. Canada. 13-16 Oct 1998. (http://lettuce.me.washin_pton.edu /micropump/puplication/l998utas98wE.htm) n a [6] Happel. J. and Brenner, H. (1983) i book: h . Rewolds rruriiber h?.drodwarirics. Martinus Nijhof Publishers, the Hague. 171 Ptiun. P . Rozenberg. G. and Salomaa, A. DNA . Conpirirrg: New Conipparirrg Paradignrs. Springes Verlag. Heidelberg. Germany. October 1998. ISBN: 3-540-64 196-3. [SI Schmidt, K., Foerster. P.. Bochmann, A. and McCaskill, J.S. (1997) A nricroflox reacforfor nro dinrerisiorial irrwstigariorts of in vitro anrplificariori s~stems. In 1" international Conference on Microreaction Technology. Book of abstracts. Dechema e.V., Frankfurt am Main. [9] Wagler, P., Gohlke, M., van Noort, D. and McCaskill. J.S. (2001j Tlzree-diniertsiorial rrricrofliridir sptenis for conrpnrufiorr ivith DNA niolecules. 12"' Micromechanics Europe Workshop MME 2001, proceedings. Cork, Ireland.

Figure 6 The romplerr design of rhu DNA-compurer f i x g,-aphs with up IO 6 irodm Tlic hluc cltmmds are rhr orqiur rirrd vqipl? ckarrnelr ,/OY rlw DNA cornpuler. erched 011 the hack qf rliu wfw. These uly con,wcred on rhr bock O rlw waj@rIO J rile otirside world, Tlrr red circles UT? rlie bend irrjecriai I d s . while rlzc rlrhr Irorironral orange clmnnels con re^ rlzc bcods,f-om the p d x IO the m c r o r siruuted bewee,! rlre WO ,~n~rrolisarior~ clrunnels. The nDbreviatior~s m e as fo1loii.s: dH is drIi?bridIsorior~inler: N is rlir neutralisation inler: Ti,, rhe input is re,nplure: Out is the o q z a from /he clique sorting ntodnles: Wa
is r1,r
i,nS!P.

The supply cliannels width on the front and the backside, are wider to reduce the flow resistance and pressurr ;sops in the complete structure. The channel width in the STMs is 100 pm. To etch through the 220 pm thick silicon wafer. etching pads on the front (400 x 400 pmj and on the back (1200 x 1200 pmj are made as to obtain holes of 400 x 400 pm, the same width as the STM-supply channels on the front of the silicon substrate.

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