Hunor Sántha
5.4 THE REALISED CASE IN THE DINAMICS PROJECT
Based on the comparison of listed lysis methods the heating of the sample to a previously determined temperature in the presence of detergent treatment was realized. This method seemed to be the most practical to be integrated into an automated system where an in-line lysis is necessary with relatively high throughput. An in-house designed electrical circuitry incorporating a microcontroller and communicating with the PC of the user via a USB link was manufactured and assembled in-house on Printed Circuit Board technology and has been linked with 2 MultiPhaserTM NE-501 programmable syringe pumps (OEM product of ProSense, Netherlands) and with an also in-house designed and manufactured lysis chamber holder and temperature actuator. A pressure controller unit is built into the system in order to decrease bubble or foam formation in the sample-buffer mix due to the heating. In Figure 5.6 the whole system is presented.
To avoid contamination single use lysis chambers made of PDMS were designed. Our preparatory calculations considering the aimed continuous flow lysis protocol, the sensitivity of the final lab-on-a-chip type detection method, the inherent losses between sample handling steps, the probable
1 1 2
3 4
Figure 5.6 Lysis control hardware 1. syringe pumps, 2. liquid handling and temperature and pressure control unit, 3. power supply, 4. lysis chamber and Falcon tube and temperature actuator unit.
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dangerous initial pathogen concentrations of water samples indicated that a lysis chamber volume of 5 ml is the optimum. Thus microfluidic channel structure has been designed with 2 inlet and 1 outlet orifices and 5 ml internal volume. The maximal outer dimensions are heights: 6 mm, widths: 72 mm, lengths: 65 mm.
Earlier a very similar pilot lysis chamber mould was used. The volume of the first version was just 1.5ml which in the light of subsequent calculation did not fit the system requirements. One of each fabricated pieces is shown in Figure 5.7.
Autodesk Inventor 2010 software was used for designing the objects. Raw PDMS was prepared by adding Sylgard 184 curing agent to Sylgard 184 silicone elastomer in 1:10 m/m ratio. The freshly prepared raw PDMS was casted in a 3D RPT fabricated mold form in a homemade casting workstation consisting of a vacuum exsiccator, a water stream based vacuum pump and tubing or in a vacuum chamber with an oil based vacuum pump. PDMS was purchased from Dow Corning Corp. (USA). For 3D RPT printing we applied an Objet Geometries (Israel) Eden 250 printer with FullCure 720 base material and FullCure 705 support material. FullCure 720 base material and FullCure 705 support material were purchased from Varinex Inc. (Hungary). During the 10 min vacuum exposition all the visible air bubbles left the PDMS body (pressure below 5 kPa). For binding two separately casted PDMS parts together we applied corona treatment surface activation with an Electro-Technic Products Inc.
BD-20AC instrument for 5 minutes. This laboratory corona treater works with three different shapes of electrodes with an output voltage between 10–48 kV and 4–5 MHz frequency range.
In the validation procedure the device was used in standalone mode thus the chambers were completed with two 60 ml syringes (sample and buffer source) and with a Falcone tube (as holder for lysate). The chamber was heated and held in its position with a fold-clamping. The mechanical parts were constructed of FR-4 plates the standard yellow base material of Printed Circuit Boards prepared of glass fibre reinforced epoxy. The temperature actuators are two Peltier units (Thermoelectric cooler, TEC) which have 50 W cooling power each and are located at either side of the chamber. The temperature on both sides was measured continuously by two sensor IC’s with at least 0.5°C accuracy (Texas Instruments TMP175). This holder is shown in Figure 5.8.
Two different softwares have been written for the lysis control hardware. Both of them are running on PC. The first one is a Graphical User Interface (GUI) and the other is an executable with parameterized inputs. The GUI is useful at the validation process and if standalone mode is used. The layout of the program window is shown in Figure 5.9. This application allows to set all process parameters
1 2
3 4
4 3
1 2
63
mm 65
Figure 5.7 The earlier version of lysis chamber (left side) and one of the fabricated pieces of the single use lysis chamber (right side) filled up with 5 ml coloured liquid (both made of PDMS). 1. heating zone, 2. mixing zone, 3. inlets, 4. outlet.
individually. The process parameters are the temperature of the lysis chamber, the pressure in the closed fluid system, the volume and the flow rate of the sample or the buffer solution. The software connects to the device automatically, additional settings are not required. The parameters to be applied in a certain lysis protocol can be pre-set by the user via a PC, and the process can be initiated by clicking on the
“START”button on the screen. After the set amount of fluid flow through the chamber into the Falcon tube the device stops automatically and steps into idle state. The process can be stopped immediately by pressing the“STOP”button at any time. The other software is created for the integrated operation.
4 2
1 3
Figure 5.8 Lysis chamber holder and temperature actuator unit (open status) 1. heating zone, 2. Falcon tube holder, 3. closing screw, 4. connector.
Figure 5.9 The Graphical User Interface.
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In the reported experiments the ratio of the lysis buffer and the sample volume were set to 1:5, namely with 10 ml/h and 50 ml/h flow respectively, thus, the residence time of any part of the liquid column subjected to the continuous lysis treatment was 5 minutes in the temperature controlled zone.
Considering the whole process the bottom and upper surface of the single use PDMS lysis chambers were heated and kept on 95°C (+2°C) and additionally at 40 kPa (+2%). Table 5.3 contains the characteristics of the control device.
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Temperature Ambient temperature 105 °C
Overpressure 0 80 kPa
Volume – 60 ml
Flow rate 1699 10−6 ml/h
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