Love Wave Fully Integrated Lab-on-chip Platform for Food Pathogen Detection - LOVE-FOOD

(Contract No 317742 – Starting Date: 1 September 2012)

Deliverable D5.1

Design and specifications of the device (μPCR + post PCR)

Due date: August 31, 2013Date of submission: October 3, 2013Author: Dr. G. Kokkoris

DELIVERABLE SUMMARY SHEET

Project Number 317742

Project Acronym LOVEFOOD

Title LOVE wave fully integrated Lab-on-Chip platform for FOOD-pathogen detection

Deliverable 5.1

Due date August 31st, 2013

Submission date October 3rd, 2013

Partners Contributed NCSR-D

Authors Dr. G. Kokkoris, Dr. A. Tserepi

Classification CO

DOCUMENT HISTORY

Date Version Description20/03/2013 1 First calculations of the temperature

distribution in a μPCR unit05/09/2013 2 Calculations on the updated design

for the μPCR unit20/09/2013 3 Revised version to coordinator

3/10/2013 3 Coordinator approves and submits to EC officer

2/12

Table of Contents

Executive Summary………………………………………………………………………..……………………………………………..4 1. Design and specifications of the μPCR unit...................................................................................5

1.1 Footprint, design, and material layers 1.2 Model for the calculations of heat transfer and fluid flow in the μPCR unit1.3 Results

2. Design and specifications of the post-PCR unit..............................................................................92.1 Footprint, design, and material layers 2.2 Model for the calculations of fluid flow and diffusion in the post-PCR unit2.3 Results

3. Conclusions…..………………………………………………………………………………………………………………………….11

3/12

Executive Summary

Need for the Deliverable

The present document describes the design of a closed-loop μPCR device and post-PCR device,

three of which will be included in the final μPCR module of the LOVE-FOOD chip. Microheaters are

integrated in the device to provide appropriate heating for the three temperature steps in PCR,

and simulations are performed to ensure the temperature uniformity in the three chambers,

necessary for efficient DNA amplification. In addition, the design of a post-PCR unit is proposed,

exploiting and only slightly modifying the existing μPCR chambers and the circulation (through the

device) of the necessary solutions for enzymatic digestion of DNA. Simulations are performed to

provide the time for complete mixing of the solutions, before digestion.

Results

According to the numerical calculations performed, a μPCR unit is designed to satisfy the

requirements for an efficient DNA amplification based on PCR: extremely uniform temperature

profile across each of the three microchambers corresponding to the three PCR temperature

steps, small power consumption, and small pressure drop across the device. For the post-PCR unit,

the same 3 microchambers with the addition of an extra one are used for the enzymatic digestion

of DNA, if single-stranded DNA detection on SAW is desired. The proposed post-PCR unit is found,

through numerical calculations, to be an efficient mixer for solutions of amplified DNA and

enzyme. A maximum of 25% increase in total PCR time is shown to be required for completion of

DNA digestion, in the same footprint of the proposed μPCR unit.

4/12

Main Text

1. Design and specifications of the μPCR module

1.1 Footprint, design, and material layers The final size of the LOVEFOOD chip is intended to be close to that of a credit-card (85x54

mm2). The bioassay strategy chosen by the consortium dictates the presence of 3 μPCR devices in the module responsible for bacterial DNA amplification. This, in combination with the envisioned area distribution among the 3 different chip modules (Deliverable 1.3, if horizontal stacking of all modules will be chosen), results into a footprint of 37x18 mm2 for each of the μPCR units. The spatial arrangement of the three basic chip modules (sample preparation, μPCR, and SAW) is designed to allow optimum thermal budget (minimized power consumption) and cross-talk (minimized interaction of heated chip zones with SAW module). A view of the whole LOVE-FOOD chip with the location of each module is shown in Fig. 1.

μPCR-unit

μPCR-unit

μPCR-unit

Figure 1. The location of the modules in the first generation LOVEFOOD chip.

The design of the μPCR unit has started during the calculation of the temperature distribution in the whole LOVE-FOOD chip (D1.3). The first version of the design is shown in Fig. 2a. Given that the heating zones of a PCR unit may affect the temperature of neighboring (unheated) regions, even only those placed a few mm away, the position of the microheaters integrated on the μPCR was chosen appropriately with respect to the two other modules (sample preparation and SAW modules). Thus, in the proposed design, the heater for the annealing, i.e. the heater for the lowest temperature, sat closer to the SAW module; the design aims to retain the operation of the SAW module unaffected from the heat sources of the μPCR units. The heater for denaturation, i.e. the heater for the highest temperature, was placed next to the sample preparation module, where heating will be anyway necessary during the cell lysis step.

In the first version of the design, the volume of denaturation, annealing, and extension chambers were chosen to be the same, appropriate for PCR protocols with time ratios of approximately 1:1:1, and equal to 4.23 μl, 4.23 μl, and 4.69 μl, respectively, summing up to a total sample volume of 13.1 μl. The height (along z direction in Fig. 2) of the chambers was 60 μm and the thickness of the channels linking the chambers was 150 μm. The second version of the design is shown in Fig. 2b and it is slightly improved compared to the first. First, the width of the microchannels connecting the chambers is increased to 500 μm. The respective increase of the

5/12

cross section of the channel is intended to reduce the pressure drop in the PCR unit, and thus loosen the requirement for high bonding strength; this increase slightly increases the volume of the chambers: The boundaries of the chambers change to adopt the increase of the channel width. Second, there is a slight shift of the extension chamber to the “top” (compare Fig. 2a with 2b). The shift of the extension chamber is intended to save space so as to include an extra chamber in the same footprint: This extra chamber is useful for the post-PCR process (see below, Section 2). Eventually, this shift and the extra chamber allow us to have both processes PCR and post-PCR taking place in the same module.

The thermal cycling in the μPCR unit will be achieved by recycling the PCR cocktail with a pump connecting inlet and outlet.

(a)

(b)

Figure 2. Top-down view of a) the first and b) the second version of the μPCR unit

The μPCR unit (and not only this but all modules of the LOVEFOOD chip) will be put on a polymer substrate [in a simplified case, on printed circuit board (PCB) substrate] with thickness of 0.76 mm. This substrate will serve as the chip assembly platform. The stack of material layers of the μPCR unit on top of the assembly platform is shown in Fig. 3.

6/12

Figure 3. yz cross section of the μPCR unit where the material and the thickness of the layers are shown. PCB substrate is not shown.

1.2 Model for the calculations of heat transfer and fluid flow in the μPCR unit

The unit cell used for the calculations of the temperature is shown in Fig. 4; it is 1/3 of the total LOVEFOOD chip containing the middle μPCR unit. The heat transfer equation in the moving DNA sample (PCR cocktail) and in the solid blocks of the unit cells as well as the momentum conservation and continuity equations in the microfluidic channel are numerically solved in 3d by a commercial code, namely COSMOL.

Figure 4. Unit cell used for the calculations of temperature distribution in the μPCR unit

It is considered that the temperature at each heater is equal to the pertinent set point, i.e. 369 K (96oC) for the heater below the denaturation chamber, 328 K (55oC) for the heater below the annealing chamber, and 348 K (75oC) for the heater below the extension chamber. Natural heat convection to the ambient (293 K) is considered for all boundary surfaces surrounded by air and periodic boundary are considered for boundary surfaces in touch with the other two μPCR units. The no slip condition is imposed to the wall of the microfluidic channel.

The outputs of the calculations are the temperature distribution in the μPCR unit, the power requirements for heating during the operation of the unit, and the pressure drop in the microfluidic channel.

7/12

1.3 Results

a) Temperature uniformity in the chambers of the μPCR unit The requirement for a high performance μPCR device is uniform fluid (DNA sample)

temperature in each one of the thermal zones (chambers in our design): The fluid temperature variation should be less than 3K in the denaturation, the annealing, and the extension chamber.The temperature distribution at the middle height of the microfluidic channel of the μPCR unit is shown in Fig. 5 for two cases of volumetric flow rate (30 μl/min and 60 μl/min). It is obvious from Fig. 5 that there is no thermal cross talk between the chambers of the μPCR unit and that the temperature uniformity is high in all chambers. A quantitative calculation verifies Fig. 5: It is calculated that more than 99% of the volume of each chamber is in the range [Tset - 1 K, Tset + 1K] (Tset is the set point for each chamber) when the volumetric flow rate is 30 μl/min. This percentage drops to 96.5% for the case of a volumetric flow rate of 60 μl/min.

(a) (b)

Figure 5. The temperature distribution on a cross section at the middle height of the microfluidic channel with the volumetric flow rate equal to a) 30 μl/min and b) 60 μl/min. The temperature values are in K.

A remark is necessary at this point: The mechanism for heat dissipation from the unit is natural convection. So, if in the final realization of the LOVEFOOD chip, any type of enclosure is used around the chip, these calculations will need to be repeated to improve the predictions.

b) The pressure drop is calculated 1505 Pa (at 30 μl/min) and 2950 Pa (at 60 μl/min). The pressure drop between the inlet and outlet of the unit is critical to prevent delamination of the cover layer sealing the μPCR device. The lower the pressure drop, the lower the bonding strength required to keep the device leak-tight.

c) The power requirements for heating during the unit operation are c.a. 1.1 W for both volumetric flow rates.

d) The time required for DNA amplification , i.e. the time for 30 cycles is c.a. 15 min (30 μl/min) and 7.5 min (60 μl/min).

8/12

9/12

2. Design and specifications of the post-PCR module

2.1 Footprint, design, and material layers In the case the SAW module requires single stranded DNA for the detection, a post-PCR

module is required for the enzymatic digestion of DNA. Restriction enzymes are used which recognize and cut DNA at specific positions. The mixing of the enzyme with the amplified DNA is important because it is the bottleneck of the digestion process. Thus, the design of an effective mixer is critical.

Two scenarios were initially considered. The first was to design a passive micromixer after the PCR unit. However, this scenario would have increased the footprint of the LOVEFOOD chip and thus was not desirable. The second scenario, which is finally followed, is to utilize for mixing the existing μPCR unit just after the DNA amplification, allowing the circulating solutions to mix. Essentially, only an extra chamber was added in the design of the μPCR unit (see Fig. 6). This chamber initially (before mixing) contains the enzyme and its buffer. Following the instructions of partner 1, the volume of this chamber is designed to be 3 μl. The extra chamber does not increase the footprint of the LOVEFOOD chip and given that the same unit will be used for both PCR and post-PCR processes, the stack of materials is the same (see section 1.1). To perform both processes, i.e. DNA amplification (PCR) and DNA digestion (post-PCR), proper valving and pumping is required. The pump is required for the recycling and the valves are required to bypass the extra chamber during the PCR process.

Figure 6. Top-down view of the post-PCR unit. The only difference with the PCR unit (see Fig. 2b) is the extra chamber for the enzyme used in the DNA digestion. A pump will be used for recycling.

2.2 Model for the calculations of fluid flow and diffusion in the post-PCR unitThe unit cell used for the calculations consists of the microfluidic channel of the post-PCR

unit shown in Fig. 6. The digestion is an isothermal process and it is considered that the whole solution is at the appropriate temperature for the digestion, i.e. at 310 K (37 oC). The mathematical problem is a diffusion-convection problem of a solute (enzyme) into water (solution of amplified DNA).

The Navier-Stokes, the continuity equation are numerically solved at steady state to calculate the velocity field. The mass balance (convection-diffusion equation) of the enzyme is

10/12

numerically solved at transient state to calculate the concentration of the enzyme. The solution of all the equations is made in 3d with a commercial code, namely COMSOL.

The outputs of the calculations are the cycles or time required for mixing and the pressure drop in the post-PCR unit.

2.3 Resultsa) Cycles or time required for mixing

Snapshots of the enzyme concentration during the recycling process are shown in Fig. 7. In particular, the enzyme concentration at the middle height of the microfluidic channel is shown. At the initial time instant, the concentration of the enzyme is high in the extra chamber (see Fig. 7a). The recycling and the successive changes of the channel cross section area contribute to mixing.

(a) (b) (c)

(d) (e) (f)Figure 7. Snapshots of enzyme concentration at the middle height of the microfluidic channel during the 1 st

cycle. The time interval between successive snapshots is 7.2 s. The diffusion coefficient is equal to 10 -10 m2/s and the volumetric flow rate is 30 μl/min.

In Fig. 8, the standard deviation of the concentration of the enzyme is shown versus the number of cycles (or equivalently the time). The calculations for a diffusion coefficient equal to 10 -

10 m2/s and a volumetric flow rate of 30 μl/min show that mixing is achieved after almost 6 cycles, i.e. after 3.5 - 4 min.

11/12

Figure 8. % standard deviation (σ) of the enzyme concentration with respect to the final enzyme concentration (Cave). The standard deviation is calculated in the whole volume of the post-PCR device.

b) The pressure drop is calculated at ~1970 Pa (30 μl/min).

3. ConclusionsFollowing the consortium decisions on the specifications of the LOVEFOOD chip and the

detection strategy, the design of the μPCR and post-PCR units has been completed. Supported by numerical calculations, the designed μPCR unit is found to satisfy the requirements for an efficient DNA amplification based on PCR: extremely uniform temperature profile across each of the three microchambers corresponding to the three PCR temperature steps, small power consumption, and small pressure drop across the device. It is planned that the same 3 microchambers with the addition of an extra one will be used for the enzymatic digestion of DNA, if single-stranded DNA detection on SAW is desired. For this reason, the proposed post-PCR unit has been tested and found to be an efficient mixer for amplified DNA and enzyme solutions. A maximum of 25% increase in total PCR time is shown to be required for completion also of DNA digestion, advantageously without changing the footprint of the μPCR module.

12/12