C - DTU Nanotech - Danmarks Tekniske Universitet

Danmarks Tekniske Universitet 

Institut for Mikro- og Nanoteknologi ∗ 

Technische Universität München 

Lehrstuhl für Bioverfahrenstechnik † 

Master Thesis 

Sampling from a Bio–Microreactor 

Sarah–Maria Fendt, B. Sc. 

Spring Term 2005 

∗ Assoc. Prof. Dr. Nicolas Szita, Assoc. Prof. Dr. Oliver Geschke 

† Univ.–Prof. Dr.–Ing. Dirk Weuster–Botz, Dipl.–Ing. Julia Hiller

Abstract 

In this thesis the feasibility to use Peltier cooling for freezing small samples and the combi- 

nation of this sampling system with a reactor chamber to compose a bio–microreactor setup 

is presented. 

In the first part of this thesis, four different sampling vessels are tested with respect to the 

efficiency of Peltier elements. It is shown that a sampling device with a laser machined 

bottom provides the best freezing times. With this device several other studies related to 

the freezing times of PBS–buffer, LB–medium and Escherichia coli cells are investigated. 

For the E. coli cells also the viability after freezing and thawing is tested. The data show 

that the freezing of samples can be conducted with a Peltier element. 

In the second part, a bio–microreactor is developed, in which the improved sampling device 

from the first part is integrated. Besides the sampling device the bio–microreactor consists 

of a reactor chamber in which the temperature is maintained with a heat wire device. The 

reactor chamber is connected through a PVC–tube interconnection and a teflon tube to the 

sampling chamber. The setup is tested with PBS–buffer. 

The data demonstrate that Peltier cooling for freezing samples and the combination with a 

reactor chamber to compose a bio–microreactor setup is suitable.

Zusammenfassung 

Diese Arbeit beschäftigt sich mit der Nutzung von Peltier Kühlung zum Einfrieren von 

Proben aus einem Bio–Mikroreaktor. 

Im ersten Teil der Arbeit wurden vier unterschiedliche Probengefäße getestet, um festzustel- 

len welches zum Einfrieren von Proben durch ein Peltier Element am geeignetsten ist. Es 

konnte gezeigt werden, dass ein Probengefäß, dessen Boden mit einem Laser bearbeitet 

wurde, die besten Gefrierzeiten aufweist. Mit diesem Gefäß wurden weitere Experimente 

bezüglich den Einfrierzeiten von PBS–Puffer, LB–Medium und E. coli Zellen durchgeführt. 

Bei den E. coli Zellen wurde zusätzlich die Überlebensrate beim Einfrieren bestimmt. Die 

Messdaten zeigen, dass Peltier Elemente zum Einfrieren von Proben grundsätzlich geeignet 

sind. 

Im zweiten Teil wurde ein Bio–Mikroreaktor entwickelt, in welchen das verbesserte Probengefäß 

vom ersten Teil integriert wurde. Neben der Probenkammer besteht der Bio–Mikroreaktor 

noch aus einer beheizbaren Reaktorkammer, die mit einer Heizdraht Komponente auf Tem- 

peratur gehalten wird. Die Reaktorkammer ist mittels einer PVC–Schlauch Kuppelung und 

einem Teflonschlauch mit dem Probengefäß verbunden. Der Bio–Mikroreaktor wurde mit 

PBS–Puffer getestet. 

Die Meßdaten zeigen, dass Peltier Kühlung für das Einfrieren von Proben aus einem Bio– 

Mikroreaktor geeignet ist.

Contents 

Nomenclature 9 

1 Introduction 11 

2 Overview of Small–Scale Cultivation Systems 13 

2.1 Shaken small–scale cultivation systems . . . . . . . . . . . . . . . . . . . . . 13 

2.2 Stirred small-scale cultivation systems . . . . . . . . . . . . . . . . . . . . . . 14 

2.3 Special small–scale cultivation systems . . . . . . . . . . . . . . . . . . . . . 14 

3 Theoretical Considerations of Polymers for Bio-Microsystems 17 

3.1 PMMA and laser micromachining . . . . . . . . . . . . . . . . . . . . . . . . 17 

3.2 Topas R○ and the CNC micromilling machine . . . . . . . . . . . . . . . . . . 19 

4 Basic Principles of a Peltier Element 23 

5 Freezing Different Samples using Peltier Cooling 27 

5.1 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 

5.1.1 Fabrication of four different freezing chambers . . . . . . . . . . . . . 27 

5.1.2 Freezing experiments with PBS–buffer relating to the freezing chamber 

design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 

5.1.3 Viability experiments with E. coli cells . . . . . . . . . . . . . . . . . 32 

5.1.4 Freezing experiments in design II with PBS–buffer and LB–medium 

relating to the volume and the temperature . . . . . . . . . . . . . . 34 

5.2 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 

5.2.1 Fabrication of four different freezing chambers . . . . . . . . . . . . . 35 

5.2.2 Freezing experiments with PBS–buffer relating to the freezing chamber 

design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 

5.2.3 Experiments with E. coli cells . . . . . . . . . . . . . . . . . . . . . . 37 

5.2.4 Freezing experiments in design II with PBS–buffer and LB–medium 

relating to the volume and the temperature . . . . . . . . . . . . . . 38 

7

8 Contents 

6 Development of a Bio–Microreactor Setup 41 

6.1 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 

6.1.1 Design of the bio–microreactor . . . . . . . . . . . . . . . . . . . . . . 42 

6.1.2 Setup of the bio–microreactor . . . . . . . . . . . . . . . . . . . . . . 55 

6.2 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 

6.2.1 Design of the bio–microreactor . . . . . . . . . . . . . . . . . . . . . . 58 

6.2.2 Setup of the bio–microreactor . . . . . . . . . . . . . . . . . . . . . . 59 

7 Concluding Remarks 63 

Appendix 67 

List of Figures 71 

List of Tables 73 

Bibliography 75

Nomenclature 

Roman symbols 

d Focal spot diameter [m] 

D Pre–focus diameter [m] 

f Focal length [m] 

It Current [A] 

t Time [s] 

T Temperature [ ◦ C] 

Tg Glass transition temperature [ ◦ C] 

Vin Incoming voltage [V] 

Vout Outgoing voltage [V] 

WP Amount of heat [W] 

Greek symbols 

α Seebeck coefficient [V/K] 

λ Wavelength [nm] 

ΠAB Peltier coefficient [V] 

Abbreviations 

AI Analog input 

AO Analog output 

CFCs Chlorofluorocarbons 

cfu Colony forming units 

CNC Computer numeric control 

9

10 Nomenclature 

COC Cyclo–olefin copolymer 

E. coli Escherichia coli 

LB Luria–Bertani 

NMR Nuclear magnetic resonance 

o/i Outer/inner 

OD Optical density 

PBS Phosphate buffered saline 

PDMS Poly (dimethyl siloxane) 

PMMA Poly (methyl methacrylate) 

PVC Poly (vinyl chloride) 

rpm Revolutions per minute 

SPR Surface plasmon resonance 

UV Ultra violet 

VIS Visible

1 Introduction 

In this thesis a bio–microreactor with a freezable sampling chamber using a Peltier element 

is presented. 

Bio–microreactors have gained increased importance in many fields of research as in the 

bioprocess development and the high–throughput screening, because of the normally low 

costs, the opportunity for parallelization and their good correlation to bench–scale bioreac- 

tors [36]. Their development has been supported by advances in online monitoring methods, 

such as optical sensors for in-situ measurements of oxygen and pH [23]. But for applications 

such as metabolite concentration or enzyme activity measurements [29], real-time measure- 

ments are difficult. Yet an efficient sampling method, which provides sampling and freezing 

of the sample in a few seconds remain important, because with this kind of sampling a lot 

of opportunities for offline measurements are provided. Fields of applications are: 

• Cell metabolism inactivation 

• Or a full automatic sampling system which keeps the samples frozen until they are 

analysed 

For cell metabolism inactivation we consider freeze–stop to be ideal, because freezing does 

not require the addition of chemicals what allows more options for the subsequent analysis of 

the sample. Furthermore a fully automatic sampling system provides a convenient possibility 

to sample during a fermentation process without consuming time and effort. For freezing 

small–scale samples Peltier elements are very dedicated, because they: 

• Respond fast 

• Are mobile applications because of their light weight 

• Are not harmful 

• And are available for micro–constructions 

Their application for freezing samples in a new developed sampling system was tested in this 

thesis. Subsequently a bio–microreactor setup was composed from the combination of the 

11

12 1 Introduction 

sampling system with a reactor chamber. 

This thesis is organized as follows: 

Chapter 2 gives a short overview over commercial available and currently developed small 

scale cultivation systems. In Chapter 3 two polymer materials, poly (methyl methacry- 

late) (PMMA) and Topas R○ , which have been used successfully for a variety of microsystems 

[27, 35], are introduced. Additionally, the machining methods for these polymers are ex- 

plained. Furthermore, in Chapter 4, the basic principle of Peltier cooling and the suitability 

of Peltier elements for different applications is described. In Chapter 5 the freezing of 

PBS–buffer, LB–medium and cell samples in a sampling chamber using a Peltier element is 

described. The chapter also includes a description of the chosen methods, the results and the 

discussion of the results. In Chapter 6, the development of a bio–microreactor with an inte- 

grated sampling system using Peltier cooling and the testing of the setup with PBS–buffer 

is presented, and the results are discussed. Chapter 7 concludes this thesis and provides an 

outlook for future work.

2 Overview of Small–Scale Cultivation 

Systems 

This chapter gives a short overview of the performance of commercial available and currently 

developed small–scale cultivation systems. 

2.1 Shaken small–scale cultivation systems 

Most of the initial culture experiments in biotechnology are performed in shaken small–scale 

systems [4]. Erlenmeyer flasks, test–tubes and microtiter plates belong to this class of shaken 

small–scale systems. 

The size of Erlenmeyer flasks is very variable, but the nominal volume normally ranges 

between 25 ml and 5 l. They are made of borosilicate glass (hydrophilic) or polymer (hy- 

drophobic) and are equipped with or without baffles. For the liquid mixing at defined 

temperatures orbital shaking devices in incubators are usually used. Dissolved oxygen can 

be measured with an integrated electrochemical O2 sensor or with a new developed opti- 

cal sensor system, which allows a robust and precise online measurement [45]. Hereby, an 

immobilized small sensor spot at the bottom of the flask is irradiated by fluorescent light 

and fluorescence quenching or decay time is sensed [23]. Because of the oxygen limitation in 

shaken flasks, various modifications like baffles and steel springs or other enhancements to 

improve aeration have been developed [5, 15, 42]. The pH is usually kept within acceptable 

range by using buffers, whereas pH excursions might occur without being observed [23]. 

But recently new developed shaken flasks with inserted pH probes have been described for 

measurement and control of pH [44, 42]. 

Test–tubes are primarily used for various screening applications and are useful for de- 

veloping inoculums for small–scale fermentations. Normally the volumes are ranging from 

about 2 − 25 ml and the tubes are made of glass or polymer. For maintaining the steril- 

ity of the culture, the opening of the tube is fitted with a cotton or a plastic foam plug. 

Test–tube devices are also used for anaerobic cultivation where the O2 concentration is kept 

below the critical values with evacuation and steel–wool plugs [37]. To use test–tubes for 

13

14 2 Overview of Small–Scale Cultivation Systems 

cultivation is very simple and cheap, but the oxygen transfer is usually low and there is no 

online–monitoring or control of pH and oxygen available [23]. 

Microtiter plates have been used for decades in medical diagnostics and later by the 

pharmaceutic industry. They offer the possibility of providing a large number of parallel 

and small reactors with identical shape. The handling can be automated using robotics with 

modern pipetting and dispensing systems, etc., so that a large number of samples can be 

handled at the same time. In the past microtiter plates have been often used for enzyme 

linked immunosorbent assays and high–throughput screening [23]. But recently microtiter 

plates are as well used for small–scale cultivation [17, 50]. The typical culture volume varies 

from 0.025 − 5 ml and standard sizes are 6, 12, 24, 48, 96, 384 and 1536 wells. Microtiter 

plates with integrated sensors for pH [40] or dissolved oxygen [17] measurements are available. 

2.2 Stirred small-scale cultivation systems 

Stirred small-scale cultivation systems are superior to most static or perfusion systems in 

terms of sampling, data collection, online–monitoring and control, and they provide a ho- 

mogeneous environment. 

Stirred minibioreactors are in their performance and monitoring very similar to conven- 

tional stirred bioreactors, except for their size. Temperature, pH and dissolved oxygen can be 

controlled. Additionally the oxygen transfer capacity is high [44]. The volume of minibiore- 

actors ranges between 50 − 300 ml. They are ideal for dedicated research projects with costly 

substrates. The primary limitation, compared to other small–scale devices, is the significant 

effort and time to operate these reactors. Due to this reason it is difficult to apply them for 

high–throughput screening. 

2.3 Special small–scale cultivation systems 

Cuvette–based minibioreactors have been developed, which employ optical sensors to suc- 

cessfully measure pH, dissolved oxygen and optical density [21]. The polystyrene cuvette 

with a total volume of 4 ml is equipped with a silicone rubber cap, which has openings for 

fresh air and exhaust air. The fermentation medium is agitated by small magnetic beads. 

The resulting pH, dissolved oxygen and optical density profiles are very similar to a parallel 

fermentation in a 1 l bioreactor [21]. 

Miniature bioreactors with integrated membrane inlet mass spectrometer probe are used 

for biological processes with advanced online analysis of volatile compounds like H2, CH4, 

O2, N2, CO2, ethanol or methanol [18]. These reactors, which are useful in analysis of 

respiratory dynamics, consist of a small, stirred reaction vessel with thermocouple, a pH

2.3 Special small–scale cultivation systems 15 

probe, agitation, temperature control and an option for aeration. The mass spectrometer is 

linked to the reactor by silicon rubber or fluorohydrogcarbon membranes, which separate the 

reactor from the high vacuum in the mass spectrometer. Selectively only volatile compounds 

are introduced into the mass spectrometer, where they are ionized and separated according to 

their mass to charge ratio. A big advantage is the continuous and sensitive detection of small 

changes in the concentration of dissolved gases, which allows fast kinetic measurements and 

in–depth metabolic studies [28, 46]. But these reactors are expensive and the measurement 

of organic volatile compounds is more difficult. 

Microbioreactors with a working volume of 5 µl and with integrated optical sensors for 

the measurement of pH, optical density and dissolved oxygen have been developed recently 

[36]. The main part of the microbioreactor is a round chamber with a diameter of 5 mm, 

which is fabricated from poly (dimethyl siloxane) (PDMS), which has a high biocompatibil- 

ity, transparency and a high gas permeability, so that it can be used as aeration membrane. 

This microbioreactor has been used in batch mode with E. coli, optical density has been 

monitored via transmittance measurements through the well of the microbioreactor, while 

dissolved oxygen and pH have been detected by using fluorescence lifetime–based sensors 

incorporated into the body of the microbioreactor [49]. Observed dissolved oxygen, optical 

density and pH profiles are similar to those of a 500 ml bench–scale bioreactor [36]. This 

small-scale cultivation system seems to be very useful for future screening applications with 

extended monitoring of cultivation profiles, however the sampling has to be optimized. 

There are a couple of other small–scale cultivation systems, like a bubble–column minibiore- 

actor [43], which is useful if the required oxygen supply is moderate and if a pH controlled 

fed–batch cultivation is needed. Or minibioreactors for online measurements of organic com- 

pounds using NMR spectroscopy [24]. Furthermore, there are specific small-scale cultivation 

systems for growing mammalian cells, for example spinner–flasks [39], tissue culture flasks 

[39], roller bottles [14] or hollow fiber based bioreactors [13, 20, 23].

3 Theoretical Considerations of 

Polymers for Bio-Microsystems 

This chapter describes the polymer materials PMMA and Topas R○ , which can be used to 

fabricate microsystems, and the ability to machine these polymers. 

3.1 PMMA and laser micromachining 

Poly (methyl methacrylate) (PMMA) is an acrylic ester polymer and it is an important 

commercial plastic in cast sheet, extruded sheet or molded parts, more known under the 

tradename Plexiglas (see Fig. 3.1). The glass transition temperature Tg of PMMA is about 

115 ◦ C [26]. The glass transition temperature is defined as the temperature at which a non– 

crystallizable polymer undergoes the transformation from a rubber to a glass [47]. PMMA, 

which is thermoplastic, can be made with almost perfect optical transmission in the range of 

360 − 1000 nm wavelength. The surface resistivity is higher than for most plastic materials, 

and the electrical properties are only affected to a minor degree by weathering or moisture. 

The high arc resistance and insulating characteristics of PMMA are important in its use in 

high-voltage circuit breakers [26, 34]. Other advantages of PMMA are: 

• Rigid 

• Good thermal shock resistance, also at deep temperatures 

• Biocompatibility 

• Resistance against weak acids and alkalis, aliphatic hydrocarbons and non–polar or- 

ganic solvents 

• Can be bonded thermally 

A disadvantage of PMMA is its chemical non-resistance against alcohol, benzol, acetone and 

polar organic solvents [33]. In addition PMMA can easily get stress cracks. 

PMMA can be machined very good with laser micromachining. Laser micromachining 

is based on the removal of polymer material by using infrared radiation or UV radiation 

17

18 3 Theoretical Considerations of Polymers for Bio-Microsystems 

CH 2 

CH3 

C 

C O 

OCH 3 

n 

Figure 3.1: Structure of PMMA [33] 

provided by a laser. Infrared laser machining evaporates substrate material directly by 

applying heat with the laser beam [35]. If UV radiation is used, the irradiated polymer 

decomposes, presumably by a mixture of two mechanisms: thermal and direct bond breaking 

[11]. As with infrared radiation, thermal bond breaking is induced by heat. In direct bond 

breaking, polymer molecules directly absorb ultraviolet photons so that the chemical bonds 

within the polymer chains will break if they absorb enough energy, which is often the case. 

The resulting smaller polymer chains are volatile or melt at much lower temperatures than 

the bulk polymer, thereby leaving a void in the material [11]. The smallest feature size 

attainable with laser machining depends strongly on the quality of the optical system, the 

laser wavelength and also the material properties of the polymer. As shown in equation 3.1 

for small feature sizes, short–wavelength radiation together with a high relative aperture of 

the optical system should be used. 

d = πλf 

4D 

d = Focal spot diameter 

λ = Wavelength 

f = Focal length 

D = Pre–focus diameter 

(3.1) 

From this the advantage of UV over infrared radiation can be seen: In practical terms 

the spot size that can be obtained with a UV system can be as small as 1.5 µm, whereas the 

feature size for an infrared laser is typically around 200 µm [2]. An advantage of the infrared 

laser is that fast prototyping is possible and it is available at moderate prices [35]. A disad- 

vantage of the infrared laser is, that the machined channels have Gaussian shape like shown 

in Fig. 3.2. For this research project only an infrared laser (CO2–laser, 1060 nm, Fig. 3.3)

100µm 

3.2 Topas R○ and the CNC micromilling machine 19 

200µm 

Figure 3.2: Profile of a channel made by an infrared laser system (the picture has been made 

by D. Snakenborg (MIC) using an optical microscope) 

and no UV laser is available. When PMMA is heated up, it remains in the solid glassy state 

until it reaches its glass transition temperature, at higher temperatures PMMA becomes 

easily rubbery and mouldable [35]. The thermal decomposition begins if more energy is 

added and results in the breaking of long polymer chains into smaller ones. For PMMA this 

process is known as depropagation, it is initiated by random chain breaking, which finally 

leads to the development of volatile methyl methacrylate and end–chain scission [1, 8, 16]. 

The main part of the depropagation occurs at temperatures between 350 − 380 ◦ C [35]. 

3.2 Topas R○ and the CNC micromilling machine 

Topas R○ is a thermoplastic cyclo–olefin copolymer (COC). The family of the cyclo–olefin 

copolymers are amorphous, transparent copolymers based on cyclo–olefins and linear olefins 

(see Fig. 3.4) and the property profile can be varied over a wide range during polymerization 

[38]. The COC Topas R○ consists of the two monomers ethylene and norbornene [27]. By 

controlling the norbornene content in the polymer, the glass transition temperature Tg can 

be engineered (Tg = 80, 130, 150, 170 ◦ C) [19]. One reason for using Topas R○ as material for 

microsystems is its transparency to light with wavelengths above 300 nm [38]. Furthermore, 

Topas R○ is chemically resistent to soap solutions, hydrolysis, acids and organic polar solvents,


Exhaust 

hood 

Marking 

head 

Sample 

holder 

CO -laser 

2 

Enclosure Power supply 

Figure 3.3: A laser micromaching system consists of a laser, a laser marking head, a computer, 

a power supply, a sample holder, an enclosure and an extractor hood. The 

laser beam enters the marking head, where the beam direction can be manipulated 

with computer control. The beam is then focussed on the sample. Any 

2.5 dimensional structure can be designed on a computer and translated into 

corresponding movements of the focussed laser beam [11])

3.2 Topas R○ and the CNC micromilling machine 21 

CH 2 

CHR 

n 

CH CH 

Figure 3.4: Structure of cyclo–olefin copolymers, consisting of cyclo–olefins and linear olefins 

[38] 

while it is soluble in non–polar organic solvents [27]. 

Topas R○ is not a good material for infrared laser machining, as it will melt instead of evap- 

orate, due to the too low power of the infrared laser. For machining Topas R○ a micromilling 

machine can be used. This technology also enables the fabrication of structures with feature 

sizes in the range of 100 µm [6]. Micromilling is a mechanical method in which a small 

revolving cutting tool removes polymer material. The position and movement of the cutting 

tool is controlled by a computer, so this process is called computer numeric control or CNC 

milling [11]. Partly because the cutting tools for micro structures tend to be fragile and 

break easily, the CNC milling is considerably slower than laser machining, due to this fact 

CNC milling is normally not used for prototyping. In comparison to infrared laser microma- 

chining, milling has the advantage that the polymer workpiece is not chemically degraded 

by heat or infrared radiation [11]. Additionally exact structures as depicted in Fig. 3.5 can 

be fabricated. 

m


100µm 

200µm 

Figure 3.5: Profile of a channel made by a CNC milling machine (the picture has been made 

by F. Bundgaard (MIC) using a scanning electron microscope)

4 Basic Principles of a Peltier Element 

In this Chapter the basic principles of Peltier cooling are illustrated. 

Peltier found in 1834 that, when an electric current passes through the junction between 

two different conductors, there is a heating or cooling effect, depending on the direction 

of the current [12]. The inversion of the Peltier effect is the Seebeck effect, that has been 

discovered in 1821: In an electric circuit of two different conductors and a temperature 

difference in the junction between them a voltage occurs at the opening of the circuit in one 

of the conductors [3]. Thus the Peltier effect is the basis of the thermoelectric refrigerator 

just as the Seebeck effect forms the basis of the thermoelectric generator [9, 12]. 

The basic principle of Peltier cooling is the transformation of electrical energy into thermal 

energy according to the following equation [25]: 

WP = ΠABIt (4.1) 

WP = Amount of heat 

ΠAB = Peltier coefficient 

I = Current 

t = Time 

For the Peltier effect, free movable electrons, which are not linked with atoms, and bonds 

lacking electrons are very important. Bonds lacking electrons behave like positive charged 

particles and are called holes. Holes are also free to move in a crystal. To built a ther- 

mocouple using the Peltier effect, a material with electrons and one with holes has to be 

connected via an electrical junction. Electrons and holes both carry positive kinetic energy. 

Energy is transported from a hole current in the same direction as the current, while an 

electron current will transport energy in the opposite direction to the current. By adjusting 

the polarity and type of current, heat can be transported to or from one side of a junction 

[22]. The Peltier coefficient ΠAB (for two materials A and B forming the junction) relates 

the current flux to the heat flux per junction. ΠAB is approximated for narrow temperature 

ranges by [22]: 

ΠAB ≈ (αA − αB)T (4.2) 

23

24 4 Basic Principles of a Peltier Element 

α = Seebeck coefficient 

T = Temperature 

Due to the heat transfer when a temperature gradient is set up across both sides of the 

junction, thermal conductivity acts to reduce the temperature gradient. This means that 

the ideal materials A and B need to have a high electrical and a low thermal conductivity. 

The high Peltier coefficient ΠAB of semiconductors enables the construction of convenient 

Peltier elements, because they have a good electrical-to-thermal conductivity ratio. The 

cooling element consists of alternating p–endowed semiconductor (Π > 0) and a n–endowed 

semiconductor (Π < 0) which are connected electrically in series by thin junctions and 

thermally in parallel between two ceramic substrates [25] (illustrated in Fig. 4.1). In the 

endowing process atoms from another element are inserted to a semiconductor crystal to 

change the conductivity of the semiconductor. These inserted charge carriers can have a 

negative charge or a positive charge, dependent on the material which is used for the endow- 

ing process. Semiconductors which are endowed with negative charge carriers are n–endowed 

and have consequently free movable electrons. These, which have been endowed with posi- 

tive charge carriers are called p–endowed and thus have holes. For further readings on the 

subject of semiconductors, I refer to [10] and also for the subject of Peltier cooling to [12]. 

The advantages of Peltier elements are their small size, the absence of wear and their silent 

running. They are often used in the following systems or because of following reasons: 

• Micro–constructions of cooling or heating systems 

• Heat transfer to hermetical closed cabinets 

• Spot cooling or heating 

• Temperature stabilization 

• Compensation of heat flow 

• Mobile application because of their light weight 

• Long–life 

• For fast and dynamic response 

• No moving parts, therefore they require little or no maintenance, ideal for cooling parts 

that may be sensitive to mechanical vibration 

• No refrigerants, such as potentially harmful CFCs, therefore environmental and safety 

benefits

p-semiconductor 

p-semiconductor 

4 Basic Principles of a Peltier Element 25 

ceramic substrate 

Current 

flow 

Hole 

flow 

Heat 

flow 

cooler side 

warmer side 

+ 

_ 

Current 

flow 

metal interconnection 

Electron 

flow 

Heat 

flow 

n-semiconductor 

n-semiconductor 

Figure 4.1: Illustration of the physical structure of a conventional Peltier effect refrigerator 

constructed from discrete p– and n–type semiconductors. In addition the polarity 

of energy and charge flows are shown [22]

5 Freezing Different Samples using 

Peltier Cooling 

This chapter describes the feasibility to use Peltier elements for freezing different samples. 

In a first test series the most suitable design of a freezing chamber for Peltier cooling was 

figured out by freezing phosphate buffered saline (PBS). As main material PMMA was 

used for the freezing chamber because of the possibility to make a rapid prototyping by 

using the CO2–laser. In subsequent experiments we determined the viability of E. coli 

after freezing the cells using Peltier cooling. The last tests engaged the freezing of different 

volumes and the temperature during the freezing of PBS–buffer. PBS–buffer was used as a 

standard, because our cell culture was diluted in PBS. While PBS–buffer was the standard, 

in addition the freezing tests with different volumes were made with Luria–Bertani–medium 

(LB–medium), because in a bio–microreactor the extracellular metabolites of the E. coli cells 

will be normally dissolved in LB–medium or a variation of the LB–medium. 

5.1 Materials and Methods 

5.1.1 Fabrication of four different freezing chambers 

The freezing chambers had a round vessel with a diameter of 8 mm and working volumes 

from 75 µl to 100 µl. By fabricating the freezing chambers, the focus was on the bottom of 

the vessel, since the Peltier element was beneath the vessel and thus a good negative heat 

transfer through the bottom of the vessel was needed. The main part of all freezing chamber 

vessels consisted of PMMA. We tested four different designs: the PMMA plate in design I 

and II was only machined with the CO2–laser (shown in Fig. 5.1). For design III and IV we 

also needed the CO2–laser, but in III, the bottom of the vessel consisted of an aluminium foil 

which was glued to a laser machined PMMA plate with vacuum grease, and for fabricating 

the bottom in IV, a PMMA foil was thermally bonded to a laser machined PMMA plate 

(see Fig. 5.1). 

Additionally two different top covers were fabricated for the freezing chambers. 

27

28 5 Freezing Different Samples using Peltier Cooling 

PMMA PMMA 

0.50 mm 0.25 mm 

PMMA PMMA 

I II 

III IV 

Aluminium foil 0.015 mm PMMA foil 0.25 mm 

Figure 5.1: Tested designs of the freezing chambers consisting of a laser machined PMMA 

plate and different bottoms 

Equipment 

• Oven Memmert 

• Vacuum grease Dow Corning 

• CO2–laser Duo Lase R○ Synrad 

• CO2–laser software WinMarkPro 4.1.1 Synrad 

• PMMA plates 100x100x2 mm 3 Nordisk Plast Danmark 

• PMMA foil 0.25 mm Röhm 

• Aluminium foil 0.015 mm Merck Eurolabs 

• Force sensor ELA–B2E–2.5KN Entran 

Method 

The CO2–laser had a wavelength of 1060 nm, a maximal power output of 65 W (equiva- 

lent 100 %) and a focal length of 190 mm. The depth of the drawn structures was defined 

through the following parameters: The power of the laser, the speed of the laser beam and 

the number of passes at each spot. Additionally the PMMA plates could be a little bit 

different in the strength of their reaction to the laser ablation. 

All the freezing chambers looked the same, except for the bottom of the vessel. The ves- 

sels and the shape of the freezing chambers were machined with the laser. For controlling

A 

B 

5.1 Materials and Methods 29 

40 mm 

26 mm 

Vessel: with a laser machined bottom 

Vessel: created as hole 

Bores for screws 

Figure 5.2: WinMarkPro drawings of the freezing chambers: (A) shows the drawing from 

design I and II, where the bottom of the vessel was fabricated with the laser. In 

the drawing from design III and IV (B) the vessel was fabricated as a hole, since 

the vessel bottom consisted of aluminium or PMMA foil 

the CO2–laser models of the freezing chambers were drawn with WinMarkPro. The laser 

settings were shown in Tab. 5.1, whereas the settings were based on experience values, such 

as the laser velocity, and on the reiteration of the machining process. The dimension of the 

laser machined PMMA plates were 20x40x2 mm 3 . Additionally the plates had two bores for 

screws, 26 mm apart from each other, and the edges were also cut (Fig. 5.2). 

A PMMA plate with an area of 20x40 mm 2 and cut edges represented the standard shape 

for thermal bonding which was used throughout this thesis. This shape was used, because 

the steel blocks, needed for thermal bonding, had an alignment apparatus for this shape. 

Design I and II 

The vessel, only machined with the laser (laser settings and WinMarkPro drawing are shown 

in Tab. 5.1 and Fig. 5.2), had in design I a depth of 1.5 mm (resulting bottom thickness: 

0.5 mm) and in II of 1.75 mm (resulting bottom thickness: 0.25 mm). The depth of the 

Shape 

20 mm


Table 5.1: CO2–laser settings for design I to IV and for the top cover 

Velocity [m/s] Power [%] Passes 

Shape 400 50 26 

I Vessel 400 30 2 

Screw bores 400 50 25 

Shape 400 50 26 

II Vessel 400 8 7 

Screw bores 400 50 25 

III Shape 400 50 26 

and Vessel 400 50 25 

IV Screw bores 400 50 25 

Top cover Shape 400 50 26 

part Vessel 400 7 7 

1 Screw bores 400 50 25 

Top cover Shape 400 50 26 

part Vessel – – – 

2 Screw bores 400 50 25 

vessels was measured with a caliper. 

Design III and IV 

In III and IV the vessel was first created as a hole in the PMMA plate by laser ablation 

(laser settings and WinMarkPro drawing are shown in Tab. 5.1 and Fig. 5.2). In III a piece 

of aluminium foil was glued with vacuum grease to the laser machined PMMA plate to form 

the bottom of the vessel. For creating a bottom in design IV, a piece of PMMA foil was 

fixed together with the laser machined PMMA plate between two steel blocks with a screw 

clamp, and was placed in an oven for 1 h at 108 ◦ C. This process is called thermal bonding. 

The pressure for thermal bonding of PMMA plates depends on the task. In a separate ex- 

periment the pressure was checked for two different tasks: To bond two plates to each other, 

and to bond small wires between two PMMA plates. The force onto the plates was measured 

with an electrical force sensor and thus the pressure was calculated. 

Top cover 

Two different top covers were fabricated for the freezing chambers. Part 1 of the top cover 

was equivalent to freezing chamber II and part 2 had the same shape as all freezing chambers 

but no vessel (laser setup is shown in Tab. 5.1). Part 1 and 2 were thermally bonded in the 

oven for 1 h at 108 ◦ C. By thermally bonding part 1 and 2 together, a top cover with an 

air gap was created. An air gap was preventing the sample, which could be loaded into the


freezing chamber, to touch the top cover (further described in section 5.2.2). Part 2 could 

be used as top cover also, but this top cover had no air gap. 

5.1.2 Freezing experiments with PBS–buffer relating to the freezing 

chamber design 

Chemicals (Vwr) 

• 10 mmol PBS–buffer 

8 g/l NaCl, 0.2 g/l KCl, 1 g/l Na2HPO4 and 0.2 g/l KH2PO4 in double distilled water 

(pH = 7.2) 

Equipment 

• Pressurized air 

• Ice 

• Cooling block 

• Freezing chambers I–IV 

• Power supply IPS2303DD Iso–Tech 

• Peltier element TEC 1–1703 Nippon India 

• Heat sink grease Circuitworks Chemtronics 

Method 

Experimental setup (Fig. 5.3): The Peltier element was placed beneath the freezing chamber, 

to cool the vessel bottom. On both sides of the element heat sink grease was used for a better 

heat conduction. The resulting heat from the Peltier element was dissipated to a cooling 

block, which was immersed in ice. The dissipation process was supported by pressurized air 

from an air gun, which flowed around the cooling block. A sample at room–temperature 

was pipetted into the vessel. In the most experiments the freezing chamber was closed with 

a top cover, which had an air gap. The top cover and the freezing chamber were screwed 

onto the cooling block, thus the Peltier element was pressed with its cool side to the freezing 

chamber and with its hot side to the cooling block. 

In the experiments with design I first no top cover, no pressurized air and no ice layer 

was used. Additionally the two top covers were tested (see section 5.1.1). The following 

experiments were performed:


PMMA 

top cover 

PMMA 

freezing 

chamber 

Sample 

Peltier 

element 

Air 

Ice 

Screw 

Heat 

sink 

grease 

Cooling block 

Figure 5.3: Experimental setup for freezing a sample using Peltier cooling. The sample drop 

is shown to touch the side wall, yet in actual experiments, different drop shapes 

and drop–to–wall interactions were observed 

• Without ice layer, pressure air, top cover 

• With ice layer and pressure air, but without top cover 

• With ice layer, pressure air, and the top cover without an air gap (part 2) 

• With ice layer, pressure air, and the top cover with an air gap (part 1 and 2) 

For the subsequent experiments with design II–IV the experimental setup with the ice layer, 

the pressure air and the top cover with an air gap was used (Fig. 5.3). 

For all experiments, 20 µl PBS–buffer were pipetted into the vessel of the freezing chamber. 

The top cover was closed and the air pressure was turned on if used. Thereafter the power 

supply was connected to the Peltier element and set to 3 A with a resulting voltage of 

about 1.1 V. The freezing time of the PBS–buffer drop was measured. This experiment was 

repeated several times for each freezing chamber. 

5.1.3 Viability experiments with E. coli cells 


• E. coli K12 



(pH = 7.2)

• LB–medium (sterile) 


5 g/l yeast extract, 5 g/l NaCl and 10 g/l peptone in double distilled water (pH = 7.5) 

• LB–plates (sterile) 

5 g/l yeast extract, 5 g/l NaCl, 10 g/l peptone and 20 mg/l agar in double distilled 

water (pH = 7.5) 

Equipment 

• Compressed air 

• Ice 


• Freezing chamber II 




• UV/Vis Photometer Ultraspec 3000 Pharmacia Biotech 

• Oven Shake ‘n‘ Stack Hybaid Limited 

• Shaker MS2 Minishaker Ika 

Method 

First 200 ml LB–medium were inoculated with the cultures from a LB–plate containing 

E. coli K12 and were placed in the oven overnight (37 ◦ C, 100 rpm). After that the OD 

was measured in an UV/VIS photometer at a wavelength of 600 nm (absorption of 0.1 

is equivalent to 10 8 cfu/ml) and the cell suspension was diluted to 10 3 cfu/ml with PBS– 

buffer. Then the same experiment as in section 5.1.2 with the same experimental setup was 

accomplished with cell culture instead of PBS–buffer in freezing chamber II. The freezing 

time was measured and the cells were kept frozen for 10 min. After the cells were thawed by 

turning off the power supply, the cell sample was streaked out on LB–plates. Additionally, 

a reference with cells, which were not frozen, was streaked out on LB–plates and the plates 

were incubated overnight at 37 ◦ C. The next day the grown colonies were counted and the 

viability of the frozen cells was calculated and compared with the reference.


5.1.4 Freezing experiments in design II with PBS–buffer and 

LB–medium relating to the volume and the temperature 


• LB–medium (sterile) 

5 g/l yeast extract, 5 g/l NaCl and 10 g/l peptone in double distilled water (pH = 7.5) 



(pH = 7.2) 

Equipment 

• Ice 


• Mercurial thermometer 

• Freezing chambers II 

• CO2–laser Duo Lase R○ SYNRAD 

• CO2–laser software WinMarkPro 4.1.1 SYNRAD 




• Temperature sensor Temperature Adapter VOLTCRAFT 

• Multimeter IDM93N Iso Tech 

• Micro–balance ISO9001 Sartorius 

Method 

The experiments were conducted with the same experimental setup as in section 5.1.2, ex- 

cept that no pressurized air was used. 

Experiments with PBS–buffer and LB–medium relating to the freezing time 

over volume

5.2 Results and discussion 35 

In the first part of the experiment, the freezing times of 2.5 µl to 20 µl PBS–buffer and 

LB-medium were measured four times. 

Due to the results from these experiments (further described in section 5.2.4) the pipettes 

were checked upon their calibration. For this 2.5 µl to 20 µl water were pipetted onto a 

micro–balance. Afterwards the experiments from above were reiterated five times with the 

checked pipettes. 

Experiments with PBS–buffer relating to the temperature over time 

In the second part, the temperature of 20 µl PBS–buffer was measured three times during 

freezing with a temperature sensor. For this a hole with a diameter of 1.36 mm was ma- 

chined into the top cover of freezing chamber II (power of the laser: 30 %, speed of the laser 

beam: 400 mm/s, number of passes at each spot: 2). The temperature sensor was immersed 

through the hole into the PBS-drop and freezing experiments as described in section 5.1.2 

were accomplished. 

Subsequently, the calibration of the temperature sensor was verified with two mercurial ther- 

mometers. For this, the temperature of a glass of tap water was measured with the sensor 

and the mercurial thermometer. 

5.2 Results and discussion 

5.2.1 Fabrication of four different freezing chambers 

The method of using the CO2–laser is standardized [35]. The bottom thickness in design I 

and II deviate about 0.05 mm depending on the used PMMA plate. 

For thermal bonding the following values were measured: The pressure, which was needed 

for bonding two PMMA plates to each other was 190 kPa, and this for bonding small wires 

between two PMMA plates was 450 kPa. 

5.2.2 Freezing experiments with PBS–buffer relating to the freezing 

Design I 

chamber design 

As described, first no ice, no pressure air and no top cover was used. With this setup, 

it was not possible to freeze the PBS–buffer. Thus the ice layer and the pressure air was 

used to obtain a better heat conduction from the cooling block to the ambience. With this 

setup, it was possible to freeze 20 µl PBS–buffer in 9.5 min. To improve this result, a top 

cover without an air gap (part 2) was used. In this configuration the problem was that


Sample 

Top cover 

Freezing chamber 

Space between top cover 

and freezing chamber 

(not to scale) 

Figure 5.4: Leaking of the freezing chamber with a top cover without an air gap, due to 

capillary forces in the interface from the freezing chamber and the top cover 

the freezing chamber was often leaking, because the top cover was only screwed onto the 

freezing chamber. Therefore, as soon as the sample touched the top cover, it was drawn out 

of the vessel by capillary forces, which arose at the interface of the top cover and the freez- 

ing chamber (Fig. 5.4). In some cases, even though the sample touched the top cover, the 

sample remained in the chamber, yet non reproducible freezing times occurred, sometimes 

with a variation of 100 %. One reason to explain this non reproducible behaviour might be 

an undefined contact area of the droplet with the top cover, thus a negative heat transfer 

through the whole system was arising. Therefore the final top cover was designed with an 

insulating air gap, situated above the drop. With this setup it was possible to freeze the 

PBS–buffer drop in exactly 2 min. 

Design II–IV 

In the experiments with design II–IV, it turned out after several reiterations that design II, 

which was only fabricated by the CO2–laser, exhibited the shortest freezing times of about 

47.38 s in average for 20 µl PBS–buffer (Fig. 5.5, data are shown in the Appendix). Also the 

deviation in design II was the smallest and the values ranged between 40 s and 50 s. The 

precision was limited, due to the fact that the freezing point was detected by visible criteria 

only, for example turbidity of the PBS drop during freezing. 

Using design III the freezing times and the deviation were higher as in design II and the 

values ranged around 60 s to 90 s with an average of 71.67 s and with a tendency to shorter 

freezing times (Fig. 5.5). So the main problem of this design was the non reproducibility 

of the data. The differences between the laser machined PMMA bottom and the PMMA 

foil, which had both the same thickness, could perhaps occur because of different PMMA

Freezing time [s] 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 


Design II Design III Design IV 

Different bio-microsystem designs 

Figure 5.5: Results of freezing PBS–buffer with Peltier cooling in different designs (the average 

of the freezing time and the min./max. deviation is plotted) 

structures, which arose by producing plates or a foil of PMMA. Another possible reason 

could be the better heat transfer through the bottom in design II because of the rougher 

surface, which resulted from laser ablation, compared to the smooth surface of a PMMA foil. 

We would have expected that design IV would have the shortest freezing times, because of 

the large heat conduction of aluminium, but it turned out that the freezing times were up to 

174 s, with a tendency to longer freezing times (Fig. 5.5). This result could not be explained 

and would require more investigation. 

5.2.3 Experiments with E. coli cells 

In these experiments it could be demonstrated that normally at least 79 % of the cells were 

alive after freezing times up to 68 s (Fig. 5.6, data are shown in the Appendix). Only one 

time, we detected the very small viability of 58.08 % after the short freezing time of 40 s, 

but most probably a mistake was made by pipetting the cell culture onto the LB-plate 

after freezing. In general, the deviation of freezing E. coli cells was higher as detected of 

PBS–buffer under the same conditions. It might be because of the different mobility of the 

cells in the culture. Or perhaps it occurred because of varieties in the cell density which 

accomplished through a non optimal mixing in the diluted cell culture.


Viability [%] 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

30 40 45 68 


Figure 5.6: Viability results of freezing E. coli cells with Peltier cooling in freezing chamber 

II 

5.2.4 Freezing experiments in design II with PBS–buffer and 

LB–medium relating to the volume and the temperature 

These experiments showed that pressure air was not needed for short freezing–thawing ex- 

periments, because the cooling block had a temperature of about 0 ◦ C during the whole 

experiment. 

Experiments with PBS–buffer and LB–medium relating to the freezing time 

over volume 

The results of the measurement of the PBS–buffer volume against the average of the freezing 

time yielded a decrease of the time (Fig. 5.7, data are shown in the Appendix). But we would 

have expected a linear relationship between the sample volume and the freezing time. One 

problem might be the exact determination of the freezing point. It could also be possible, 

that the calibration of the pipettes was not exact. Also the deviation at 10 µl PBS–buffer to 

higher values and LB–medium to lower values might occur because of different pipettes since 

the experiments were not conducted at the same day. To determine this, the calibration of 

the pipettes was checked and the experiments were reiterated. This reiteration was per- 

formed with a single pipette, which was proven to be well calibrated. The deviation over the 

whole pipetting volume was only ±1 %. As a result of this reiteration the measuring data for 

PBS–buffer and LB-medium showed an approximately linear decrease with almost the same 

slope, but exhibited moderate standard deviations (shown in Fig. 5.8, data are shown in the


50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 


PBS-buffer LB-medium 

0 5 10 15 20 25 

Volume [µl] 

Figure 5.7: Averages of the freezing times against the different volumes of PBS–buffer and 

LB–medium. The standard deviation is shown 

Appendix). Due to this result, it can be concluded that in the former experiments pipettes 

were used whose calibration was incorrect. The absolute values of the freezing times were 

different, because of the different parameters of PBS–buffer and LB–medium, for example 

the salt concentration. Therefore, it can be assumed that the freezing time decreased linear 

with the sample volume. 

Experiments with PBS–buffer relating to the temperature over time 

The temperature in a PBS–drop was measured during freezing and the experiment was 

repeated three times. As expected the resulting curves had a plateau during the phase tran- 

sition. The curve of the first and the third experiment were very much the same, except for 

the end temperature: in the first experiment the temperature was −10.9 ◦ C, in the third ex- 

periment −9.1 ◦ C after 4 min. In the second experiment, the temperature in the PBS–buffer 

drop reached the 0 ◦ C 30 s later as in the other two experiments. The end temperature was 

−9.2 ◦ C (Fig. 5.9, data are shown in the Appendix). The absolute temperatures were not 

so important, due to the hole in the top cover a negative heat transfer occurred and the 

metal temperature sensor was also conducting heat out of the system. So the experiments 

showed that the Peltier element was working stable but not 100 % reproducible. A minimal 

temperature of −11.4 ◦ C was measured after 10 min of freezing. 

Verifying the thermometer accuracy, the results showed a difference of about 1 ◦ C between 

the two mercurial thermometers and the VOLTCRAFT temperature sensor. Most probably 

the values from the sensor were about 1 ◦ C too high.



50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

PBS-buffer LB-medium 

0 5 10 15 20 25 

Volume [µl] 

Figure 5.8: Averages of the freezing times against the different volumes of PBS–buffer and 

LB–medium from the reiterated experiment with a well calibrated pipette. The 

standard deviation is shown 

Temperatur [°C] 

25 

20 

15 

10 

5 

0 

-5 

-10 

-15 

0 50 100 150 200 250 300 

Time [min] 

Figure 5.9: Temperatures of 20 µl PBS–buffer during the freezing using a Peltier element

6 Development of a Bio–Microreactor 

Setup 

In this chapter, the further development of the freezing chamber from Chapter 5 to a bio– 

microreactor is described. In a second step, the single parts of the bio–microreactor setup 

were tested. And subsequently all parts were connected together and the bio–microreactor 

was tested with PBS–buffer. 

The bio–microreactor setup consisted of two main parts made of PMMA: The reactor 

chamber where a fermentation can take place, and a sampling chamber in which the sample 

will be frozen by using a Peltier element. The basic structure of the sampling chamber 

was equivalent to the freezing chamber introduced in Chapter 5. To provide flexibility the 

bio–microreactor was developed as a plug in system consisting of different devices. This will 

allow the option to interconnect an extra part between the reactor chamber and the sampling 

chamber, for example a surface plasmon resonance (SPR) system for measuring metabolite 

or protein concentrations semi–online. 

The reactor chamber had a working volume of 225 µl and was machined mainly from PMMA. 

For aeration, a thin poly (dimethyl siloxane) (PDMS) membrane on the top of the vessel 

was used [49]. The temperature was controlled with a temperature sensor and an electrical 

resistance heat device [31, 32]. For sampling 10 µl of the solution in the reactor chamber 

could be pumped with a syringe pump to the sampling chamber (Fig. 6.1 I and II). The 

sampling chamber was built like the freezing chamber design II presented in Chapter 5, with 

the exception that it had an inlet and an outlet for sampling. And additionally, a sticky tape 

was used to close the vessel to the air gap in the top cover (shown in Fig. 6.1). The sticky 

tape was used to prevent leaking, and the air gap in the top cover was fabricated to have 

a better thermal insulation. For freezing samples in the sampling chamber, a new Peltier 

element with higher power than that of Chapter 5 was used. To provide the heat conduction 

from the Peltier element, a liquid heat exchanger was used, instead of a cooling block as in 

Chapter 5. 

41

42 6 Development of a Bio–Microreactor Setup 

Reactor chamber 

I PDMS membrane 

II 

Heat device 

Cell culture 


PDMS membrane 

Heat device 

Cell culture 

Top 

cover 

Top 

cover 

Sampling chamber 

Peltier element 

with liquid heat exchanger 




Sticky 

tape 

Sticky 

tape 

Syringe pump 

Syringe pump 

Figure 6.1: Setup of the bio–microreactor in two different conditions: I shows the conditions 

in the reactor chamber before sampling. In II the sampling is pictured 

6.1 Materials and Methods 

6.1.1 Design of the bio–microreactor 

Chemicals 

• Silastic M Kit Milastic Dow Corning 

• Sylgard 184 Kit Silicon elastomer Dow Corning 

Equipment 

• Pressure air 

• Epoxy Kit Rsg 

• Conductive Epoxy Kit Circuitworks 

• Oven Memmert 

• Desiccator Bel–Art


• Drilling machine 16 BS Klee 

• CO2–laser Duo Lase R○ Synrad 

• CO2–laser software WinMarkPro 4.1.1 Synrad 

• PMMA plates 100x100x1.5 mm 3 Nordisk Plast Danmark 

• PMMA foil 0.25 mm Röhm 

• Sticky tape Heat resistant Scotch 

• Heater ITO PE film 0.20 mm Sigma Aldrich 

• Temperature sensor 256–045 Rs–components 

• Spin coater Spin 150 APT 

• PVC–tube o/i–ø 2.3/0.8 mm Vwr 

• Heat wire WSD-1 Monacor 

• Profilometer Dektak 8 Veeco 

Method 

For designing the bio–microreactor, the interconnections, the reactor chamber with the heat 

device and the sampling chamber with the Peltier element had to be built. 

Interconnections 

Since the bio–microreactor was developed as a plug in system, interconnections between the 

different parts were very important. 

Silastic M O–rings 

O–ring interconnections consisted of a Silastic M O–ring and a small metal capillary. To 

integrate an O–ring into a channel, a cavity was made into a channel and the O–ring was 

thermally bonded between two PMMA plates (Fig. 6.2). A capillary could be plugged into 

the channel through the O–rings and leaking could be prevented this way. 

For producing the O–ring with an inner diameter of 0.24 mm, a maximum outer diameter of 

2.0 mm and a height of 2.0 mm, a mold was made with the CO2–laser in PMMA. The mold 

consisted of two equivalent parts which were bonded together thermally. The dimensions of 

one part were 20x40x1.5 mm 3 . The standard shape for thermal bonding was used (section 

5.1.1). For producing the mold a circular structure (consisting of eight circles) with injection


Capillary 

O-ring 

Channel 

PMMA plate 

Figure 6.2: O–ring interconnection in a channel [30] 

Injection channel Shape 

Circles 

Figure 6.3: WinMarkPro drawing of the mold for the O–rings (not to scale) 

Injection channel 

Cavity 

PMMA plate 

Figure 6.4: Longitudinal section of the mold for O–rings


channels was drawn in WinMarkPro (shown in Fig. 6.3). Because the depth of laser ma- 

chined structures was defined through the laser settings, three different settings were tested 

for producing the height of the O–rings, shown in Tab. 6.1. Subsequently, the two laser 

machined parts of the mold were bonded thermally in the oven for 1 h at 108 ◦ C (Fig. 6.4). 

For producing O–rings the Sicastic M Kit was prepared: The curing agent was mixed with 

silicone polymer in the ratio 1:10. The mixture was placed into a desiccator for 1 h to re- 

move air bubbles. Subsequently the mixture was injected with a needle into the mold and 

the injected mold was placed for the curing process into the oven for 45 min at 108 ◦ C. 

Due to the too large flexibility of the resulting O–rings the whole fabrication process was 

reiterated with new laser settings as shown in Tab. 6.2. The settings were changed to obtain 

O–rings with a thicker wall. For this the ablation thickness in circle 8 was increased with a 

function called wobbling and also the drawing of two more circles was tested. 

PVC–tube interconnections 

For producing poly(vinyl chloride) (PVC)–tube interconnections, the device depicted in 

Fig. 6.5 was fabricated. In a first step, a hole was ablated with the laser into two PMMA 

plates building a vessel. Thereafter, the two PMMA plates and an additional plate forming 

the bottom were aligned with thermal resistant sticky tape. The small bore (with a diame- 

ter of 1.1 mm) and the large bore (with a diameter of 2.3 mm and a depth of 3.0 mm) were 

fabricated according to Fig. 6.5. For cooling during drilling soap–water was used instead of 

coolant, because coolant disturbs bonding. The plates were dried after drilling with pressur- 

ized air. Subsequently, a PVC–tube with an outer diameter of 2.3 mm, an inner diameter of 

0.8 mm and a length of 7 mm was plugged into the large bore, and the plates were thermally 

bonded at 108 ◦ C for 1 h. Afterwards, the interconnection was tested for leaking by injecting 

water with a syringe through the PVC–tube into the vessel. 


For the reactor chamber, the standard shape for bonding (section 5.1.1), including bores for 

screws, was used. 

One step bonding 

The chamber consisted of several parts (Fig. 6.6). The laser settings were shown in Tab. 6.3. 

The vessel consisted of three parts made out of PMMA. In these PMMA plates, holes were 

machined with a diameter of 8 mm using the laser. Additionally, in plate II and III, a chan- 

nel for a PVC–tube interconnection was drilled as described above. The interconnection to 

the sampling chamber led centric to the vessel. Beneath the PMMA plates, which built the


Table 6.1: Three different CO2–laser settings for the O–ring mold (circles shown in Fig. 6.3) 

Radius [mm] Velocity [m/s] Power [%] Passes 

Circle 1 0.12 400 7 4 

Circle 2 0.25 400 7 4 

Circle 3 0.38 400 10 4 

Circle 4 0.50 400 50 4 

Circle 5 0.62 400 50 4 

Circle 6 0.75 400 50 4 

Circle 7 0.88 400 70 4 

Circle 8 1.00 400 70 4 

Channels – 400 25 2 

Shape – 400 50 25 

Circle 1 0.12 400 4 7 

Circle 2 0.25 400 4 7 

Circle 3 0.38 400 5 8 

Circle 4 0.50 400 25 8 

Circle 5 0.62 400 25 8 

Circle 6 0.75 400 25 8 

Circle 7 0.88 400 35 8 

Circle 8 1.00 400 35 8 


Shape – 400 50 25 

Circle 1 0.12 400 2 13 

Circle 2 0.25 400 2 13 

Circle 3 0.38 400 3 13 

Circle 4 0.50 400 17 11 

Circle 5 0.62 400 17 11 

Circle 6 0.75 400 17 11 

Circle 7 0.88 400 23 13 

Circle 8 1.00 400 23 13 


Shape – 400 50 25


Table 6.2: Three changes in the CO2–laser settings for the O–ring mold. The basic settings 

are the same as in the first part of Tab. 6.1. Different are the settings for circle 8 

and in the last setting two new circles were drawn 

Radius [mm] Velocity [m/s] Power [%] Passes Wobbling [mm] 

Circle 1 0.12 400 7 4 – 

Circle 2 0.25 400 7 4 – 

Circle 3 0.38 400 10 4 – 

Circle 4 0.50 400 50 4 – 

Circle 5 0.62 400 50 4 – 

Circle 6 0.75 400 50 4 – 

Circle 7 0.88 400 70 4 – 

Circle 8 1.00 400 10 1 0.15 

Channels – 400 25 2 – 

Shape – 400 50 25 – 

Circle 1 0.12 400 7 4 – 

Circle 2 0.25 400 7 4 – 

Circle 3 0.38 400 10 4 – 

Circle 4 0.50 400 50 4 – 

Circle 5 0.62 400 50 4 – 

Circle 6 0.75 400 50 4 – 

Circle 7 0.88 400 70 4 – 

Circle 8 1.00 400 10 1 0.20 

Channels – 400 25 2 – 

Shape – 400 50 25 – 

Circle 1 0.12 400 7 4 – 

Circle 2 0.25 400 7 4 – 

Circle 3 0.38 400 10 4 – 

Circle 4 0.50 400 50 4 – 

Circle 5 0.62 400 50 4 – 

Circle 6 0.75 400 50 4 – 

Circle 7 0.88 400 70 4 – 

Circle 8 1.00 400 30 4 – 

Circle 9 1.05 400 20 1 – 

Circle 10 1.10 400 10 1 – 

Channels – 400 25 2 – 

Shape – 400 50 25 –


A 

PMMA plates 

B 

C 

Vessel Small bore Large bore 

PVC-tube PVC-tube 

Teflon-tube 

Figure 6.5: Cross section (A) of the bores for the PVC–tube interconnections, from a device, 

such as depicted in (B). In (C) two PVC–tube interconnections, connected with 

a teflon–tube are depicted

I 

II 

III 

IV 

V 

VI 


Figure 6.6: First reactor chamber setup: PMMA plates I–III built the reactor vessel, II and 

III had a channel with PVC–tube interconnection for sampling. A temperature 

sensor (not shown) was bonded in between III and the PMMA foil IV. Beneath 

IV was the heat foil (V) and the final reactor chamber bottom (VI) with cut offs 

for the heat foil and the electric wires (not shown)


Table 6.3: CO2–laser settings for the reactor chamber 


For Shape 400 50 25 

PMMA Vessel (if needed) 400 50 25 

plates Screw bores 400 50 25 


PMMA Vessel 400 50 1 

foil Screw bores 400 50 1 

Bottom Cut off: heat foil 400 9 1 

plate Cut off: wires 400 48 1 

vessel, a temperature sensor passed into the reactor vessel. The vessel bottom was a 250 µm 

thin PMMA foil (IV), beneath this was the heat foil (V) and the final reactor chamber bot- 

tom. In the final PMMA plate (VI) were cut offs for the heat foil (19x12x0.2 mm 3 ) and the 

electric wires. With a conductive epoxy kit (mixing ratio of part A and B of the kit was 

1:1) the electric contact wires were glued to the conductive side of the heat foil and thereby 

also to the final PMMA plate. The curing process took around 20 min at room temperature. 

Because the heat foil did not bond directly to PMMA, the thin PMMA foil (IV) was used to 

create the bottom of the reactor. Subsequently, all different layers were thermally bonded 

at 108 ◦ C for 1 h: 

• Reactor vessel, consisting of PMMA plate I, plate II and III containing the intercon- 

nection 

• Temperature sensor 

• PMMA foil for the vessel bottom 

• PMMA plate for the reactor chamber bottom, with the electric contact wires and the 

heat foil glued onto it 

To avoid short circuiting of the wires of the temperature sensor, the wires were insulated 

with non conductive epoxy. The epoxy kit was mixed in the ratio 1:1, and a drop of the 

mixture was placed on the temperature sensor and the wires. This was subsequently cured 

for 15 min at room temperature. 

Bonding and screwing 

Due to the problems occurring during the fabrication process (described in detail in section 

6.2.1), the chamber was made another way (laser settings are shown in Tab. 6.4). The reactor 

vessel was built the same way as described above, with the exception that the temperature


Table 6.4: New CO2–laser settings for the reactor chamber 



PMMA Vessel (if needed) 400 50 25 

plates Screw bores 400 50 25 



foil Screw bores 400 50 1 

Middle Cut off: Temperature sensor 400 8 5 


Bottom Cut off: heat foil 400 9 1 


sensor was not passing into the reactor vessel. The four layers and the PVC–tubes for the 

interconnections were thermally bonded at 108 ◦ C for 1 h as shown in Fig. 6.7. For the tem- 

perature sensor (4.6x1.7x1.3 mm 3 ) and its electric wires cut offs were made in a new PMMA 

plate. The plate for the heat foil and the preparation of the heat foil and its wires was 

made as described above. The plate for the temperature sensor with the sensor was screwed 

between the bonded parts building the vessel and the plate with the heat foil (Fig. 6.7). 

Heating the chamber 

Due to the results described in section 6.2.2 an electrical resistance heat wire device was used 

[31]. The heat wire device was made by Sarunas Petronis (MIC). Therefore line cavities with 

a thickness of 0.3 mm, a depth of around 0.3 mm and a space between each line of 1 mm 

were made with the laser into PMMA (laser settings: velocity 400 mm/s, power 10 % and 

number of passes 1). Subsequently, another plate with no cavities was thermally bonded at 

108 ◦ C for 1 h to the plate with cavities (Fig. 6.8). Thereafter, the heat wire was immersed 

through the cavities. For building the reactor chamber, the vessel as described above was 

glued with sticky tape to the PMMA plate containing the temperature sensor. This was 

thereafter stuck to the PMMA heat wire device. 

Closing the chamber 

The chamber was closed with a thin PDMS membrane with a thickness of around 40 µm [49]. 

The membrane prevented dehydration and contaminations. Aeration through the membrane 

was possible. The thin PDMS membrane was made with a spin coater. Primarily the PDMS 

curing agent was mixed with silicone polymer in the ratio 1:10. The mixture was placed into 

a desiccator for 1 h to remove air bubbles. Subsequently, 1 ml of the mixture was placed 

with a syringe onto a PMMA plate which was fixed in the spin coater by using vacuum. The


I 

II 

III 

IV 

V 

VI 

Figure 6.7: Second reactor chamber setup: PMMA plates I–IV built the reactor vessel, II and 

III had a channel with a PVC–tube interconnection for sampling and the PMMA 

foil IV was closing the vessel. These parts were thermally bonded together. The 

temperature sensor (not shown) was placed into the PMMA plate V and the 

PMMA plate VI contains the heat foil (not shown) and the electric wires (not 

shown). All parts were screwed together

I 

II 


Figure 6.8: Heat wire device (not to scale): The two PMMA plates I and II were bonded 

thermally together so that the heat wire (not shown) was immersed through the 

cavities 

spin coater had an acceleration of 400 rpm/s. In the first step it rotated 10 s at 400 rpm and 

in the second step 40 s at 1500 rpm. Afterwards, the PDMS coated plates were placed for 

curing into the oven at 70 ◦ C for 1 h. The thickness of one of the produced membranes was 

measured with a profilometer. Finally the PDMS membrane was cut into pieces and could 

be placed onto the reactor chamber as a top cover. 


The sampling chambers consisted of two main parts, the top cover and the sampling vessel 

with a diameter of 4 mm, 3 mm or 2 mm. (Fig. 6.9). The PMMA plates were laser machined 

like the freezing chamber design II in section 5.1.1 (laser settings are shown in Tab. 6.5). 

Additionally two channels for sampling with PVC–tube interconnections (one to the reactor 

chamber and the other to the syringe pump) were drilled as described above for the PVC– 

tube interconnections. A sticky tape was sealing the chambers to the air gap in the top 

cover. For each sampling chamber, a top cover was made the same way as the top cover 

in section 5.1.1. The air gaps had the same diameter as the sampling chamber vessels. All 

three sampling chambers were tested.


I 

II 

III 

IV 

Figure 6.9: The sampling chamber consisted of two main parts, the top cover (PMMA plates 

I and II were thermally bonded together) and the sampling vessel with PVC–tube 

interconnections for sampling (PMMA plates III and IV were thermally bonded 

together). The sampling vessel was closed with a sticky tape (not shown)


Table 6.5: CO2–laser settings for the three sampling chambers 


Upper Shape 400 50 25 


Vessel with a plate Screw bores 400 50 25 

diameter of 4 mm Lower Shape 400 50 25 


plate Screw bores 400 50 25 













6.1.2 Setup of the bio–microreactor 




(pH = 7.2) 

Equipment 

• Reactor chamber 

• Sampling chamber 

• Syringes 1010 Gastight 

• Fitting P 295 Upchurch Scientific 

Upchurch Scientific 

• Teflon–tube o/i–ø 2.2/1.0 mm Bohlender 

• Syringe pumps PHD 2000 Harvard Apperatus


• I/O card LabJack U 12 Meilenhaus Electronic 

• LabView 7.0 NationalInstruments 

• Aluminium plate 51x57x4 mm 3 Workshop (Mic) 

• Liquid heat exchanger Li-201TH-M amsTechnologies 

• Peltier element CPO.8-71-06L amsTechnologies 



Method 

The reactor chamber was connected with a teflon–tube to the sampling chamber. For this, 

the 3 cm long teflon–tube was cut at both ends angular and was immersed into the PVC– 

tubes from the interconnection at the sampling chamber and the reactor chamber. From the 

sampling chamber another teflon–tube with a length of 30 cm led via a fitting to a syringe 

pump. The temperature sensor and the electric wires from the heat foil were connected 

via an electronic circuit to a LabJack I/O card (Fig. 6.10). The heat foil was controlled 

by a LabView program, written by Sarunas Petronis (MIC), which used the data from the 

temperature sensor. The temperature sensor was calibrated by filling the reactor chamber 

with water of different temperatures. 

For conducting the heat away from the Peltier element at the sampling chamber a liquid 

heat exchanger was used. Because the bores in the liquid heat exchanger did not fit with the 

bores in the sampling chamber, an aluminium plate was fabricated. The interface between 

the liquid heat exchanger and the aluminium plate was coated with a thin layer of heat sink 

grease and the aluminium plate was screwed onto the liquid heat exchanger. Between the 

aluminium plate and the Peltier element, and between the Peltier element and the sampling 

chamber also a thin layer of heat sink grease was placed. The sampling chamber with the 

lid was screwed onto the aluminium plate and the Peltier element was connected to a power 

supply. 

Due to the problems, which occurred during the testing of the system (section 6.2.2) in the 

final setup the heat wire device was used instead of the heat foil. 

Subsequently, the setup was tested with PBS–buffer. First the reactor chamber was filled 

with the maximal volume of 225 µl PBS–buffer. The chamber was closed with the thin PDMS 

membrane. The LabView program controlling the temperature through the temperature 

sensor and the heat wire device was set to 37 ◦ C. First the LabView program controlling the

I 

II 

V in 

(LabJack AO) 

_ 

+ 

LM 324N 

+ 

_ 

75kΩ 75kΩ 

30kΩ 

LM 324N 


TIP 120 

2N2907 _ 

10kΩ 

Thermistor 

+ 9-12 V (power source) 

V out (heater) 

Figure 6.10: Electronic circuit to interface USB LabJack U12 data acquisition and control 

card with the heater and thermistor. Scheme I was designed to control the 

Fig. 2. Electronic circuits to interface USB LabJack U12 data acquisition and control 

electrical power provided to the heater by the voltage Vin level in the analog 

card with the heater and thermistor. Scheme (a) is designed to control the electrical 

output (AO) port of the LabJack. The voltage Vout applied on the heater follows 

power provided to the heater by the voltage Vin level in the analogical output (AO) 

Vin but with much higher current supplied from the external power source. 

port of the LabJack. The voltage Vout applied on the heater follows Vin but with much 

Scheme II keeps constant current (50 µA) passing through the thermistor into 

higher current supplied from the external power source. Scheme (b) keeps constant 

voltage registered by the analog input (AI) port of the LabJack (drawing made 

current (50 µA) passing through the thermistor and therefore converts temperature 

by Sarunas Petronis (MIC) 

dependant resistance changes of the thermistor into voltage registered by the 

analogical input (AI) port of the LabJack. 

+ 

LM 324N 

+ 5 V 

V out 

(LabJack AI)


temperature was started. After reaching 37 ◦ C the Peltier element was sarted to pre–cool 

the sampling chamber, by turning on the power supply (6.1 V, 1.3 A). With the syringe 

pump 150 µl were pumped with a flow rate of 5 ml/min from the reactor chamber through 

the sampling chamber into the teflon tube leading to the syringe. Thereby, the sampling 

chamber was filled with around 15.7 µl, 8.8 µl or 3.9 µl, depending on the sampling chamber 

which was used and was frozen by the Peltier element. Due to gas fractions in the tube 

leading to the syringe pump and in the syringes, a time delay between starting the pump 

and starting of the fluid flow occurred. In a separate experiment the top cover and the sticky 

tape were removed to gain some knowledge about the fill level in the chamber. 

6.2 Results and discussion 

6.2.1 Design of the bio–microreactor 

Interconnections 

O–ring interconnections 

In the laser settings two and three from Tab. 6.1 problems in the alignment occurred. The 

O–rings had no hole in the middle. One reason for the bad alignment could be that the 

pressure induced by fixing the two parts of the mold with screw clamps during bonding 

was too low. Another possible reason were the laser settings themselves. By ablating the 

smallest circle with different laser power and number of passes, the tips of the cones in the 

two parts of the mold could perhaps been abbreviated, so that they could not touch each 

other during the bonding process. 

Only the O–rings from the first setting were complete O–rings. The problem that occurred 

with these O–rings was that they would not maintain shape while bonding them between 

two PMMA plates. This was due to their thin walls. 

As described in section 6.1.1, new laser settings were created to obtain a thicker wall of 

the O–ring. But the results by using the wobble function were almost the same as before. 

Probably the wobble function had not worked, which could be a problem of the software. 

By drawing more circles only the cone turned out to become bigger, which led to no changes 

in the wall thickness. To optimize the wall thickness, the solution with more circles might 

work, if the laser power and the number of passes was changed to ablate less PMMA. But 

we decided, that the optimization process will take too much time. Therefore, we focussed 

on the solution with PVC–tubes as described in section 6.1.1. 

PVC–tube interconnections 

The PVC tube interconnections offered a good feasibility to prevent leaking at the connec-


tion between two parts. Additionally, plugging in and out was possible several times (around 

twenty times) before the PVC–tube was breaking out of the bonded PMMA plates or the 

PVC–tube was too widened so that it was not longer surrounding the teflon–tube with the 

needed strength. It can be concluded that PVC tubes were an easy and cheap way to make 

plug in interconnections. 


Building the reactor chamber, a problem occurred while bonding the temperature sensor into 

the reactor vessel. If the pressure imposed by the screw clamps during bonding was too high 

(around 450 kPa), the channels for mixing and sampling melted. If the pressure was set to 

a value (around 190 kPa) where no deformation took place, the bonding was not successful 

and the reactor was leaking. As solution the temperature sensor was placed directly under 

the vessel, this had also the advantage that it was reusable if the vessel had to be changed. 

The thickness of one of the PDMS membranes, measured with a profilometer, was 40 µm. 


Fabricating the sampling chambers no problems occurred. 

6.2.2 Setup of the bio–microreactor 

During the setup of the bio–microreactor no problems occurred. An annotated photograph 

of the bio–microreactor is shown in Fig. 6.11. 

During testing the setup several problems occurred. By closing the reactor chamber with 

the thin PDMS membrane it had to be taken care, that the chamber was not leaking. For 

this purpose, the reactor had to be filled very accurate and the chamber had to be closed 

very carefully. Additionally there was a sterilization problem, because by closing the reactor 

the thin PDMS membrane was touched with hands. Another problem was that the heat foil 

burned several times by connecting it to the power source. This happened also at a very 

low current. The problem might be the fabrication, because the foil was very sensitive. So 

we decided to use the heat wire device instead, which had worked very well. To reach 37 ◦ C 

in the reactor, the program was set to a value of 38 ◦ C and a voltage of 4 V was needed. 

This setting had to be applied, because the regulation from the LabView program was 

lowering the voltage before reaching the desired temperature. In the sampling experiments 

rapid freezing was achieved using the Peltier element which was turned on around 2 min 

before sampling. In five sampling experiments for each sampling chamber the data shown in 

Tab. 6.6 were measured. The results showed that for sampling and freezing of PBS–buffer 

samples the same time was needed in almost every experiment. Only in one experiment for


Sampling vessel 


Teflon- tube 

leading to the 

syringe pump 

Wires leading 

to the Peltier 

element 

Sampling 

chamber 

Liquid heat 

exchanger 

Peltier 

element 

Teflon-tube 

between 

the sampling 

and the 

reactor chamber 

Reactor 

chamber 

Wires from the 

heat wire device 


LabJack card 

Figure 6.11: Photograph of the bio–microreactor 

Reactor vessel 

Wires from the 

temperature sensor 


LabJack card 

Temperature sensor 

PVC-interconnection 

with teflon-tube 

Heat wire device


Table 6.6: Measured data for freezing PBS–buffer samples 

Time delay [s] Pumping time [s] Freezing time [s] 

Sampling Sample 1 1 2 6 

chamber Sample 2 1 2 6 

with a Sample 3 1 2 7 

volume of Sample 4 1 2 6 

15.7 µl Sample 5 1 2 6 





8.8 µl Sample 5 1 2 4 





3.9 µl Sample 5 1 2 3 

each sampling chamber it lasted 1 s longer, which could be a statistic deviation. The time 

delay could be neglected, because pumping out had not started and the conditions in the 

reactor chamber had not changed yet. The freezing was detected by visual inspection and 

by trying to pump the sample back to the reactor. The freezing times from each sampling 

chamber are shown in Fig. 6.12. The graph shows an almost straight line, but the freezing 

time for 3.9 µl was too high. This might be, because the filling volume could not be measured 

exactly under frozen conditions and under thawed conditions the liquid in the channels and 

the interconnections adulterated the result. It turned out that the surface of the sample in 

the smaller sampling chambers was arced rather than flat. But the shape of the surface and 

the filling was only detected by visible criteria. Additionally the bottom of the chambers was 

only approximately 0.25 mm thick, because of the laser and the PMMA plates as described 

in section 5.1.1. Because of these reasons only the estimated volumes, defined through the 

geometry of the sampling chamber, were used to plot the graph in Fig. 6.12.



7 

6 

5 

4 

3 

2 

1 

0 

0 5 10 15 20 

Volume [µl] 

Figure 6.12: Average of the freezing time against the different volumes of the sampling chambers. 

The standard deviation and a linear trendline is shown

7 Concluding Remarks 

In this thesis, the feasibility to use Peltier cooling for freezing small samples and the combi- 

nation of this sampling system with a reactor chamber to compose a bio–microreactor setup 

was discussed. 

In the first part of this thesis, it could be demonstrated that a laser machined bottom in 

the freezing chamber allowed the best negative heat transfer from all tested designs. In this 

freezing chamber design E. coli K12 cells were frozen. The viability of the cells was tested 

after thawing, by streaking them out onto LB–plates and performing a colony count, after 

the plates were kept for one night at 37 ◦ C. The achieved viability was generally higher than 

79 % at average freezing times of 45.75 s, but the deviation was larger than with PBS–buffer. 

The viability test provided only the information whether the cells were healthy enough to 

divide themselves again and to build a colony, but it gave no information about the integrity 

of the cell membrane. The integrity of the cell membrane is of particular importance, if the 

focus is on intracellular metabolites. To check the integrity of the membrane, a dead/alive 

stain would be required. Furthermore, the data from freezing different volumes of PBS– 

buffer and LB–media were showing, that the freezing time yielded a linear relationship with 

the sample volume. These observations led us to develop a bio–microreactor setup, com- 

posed of a sampling chamber, where the sample could be frozen using Peltier cooling and a 

reactor chamber in which temperature control was possible. 

The final setup of the bio–microreactor setup is shown in Fig. 7.1. This final setup had 

PVC–tube interconnections built into the sampling chamber and the reactor chamber. A 

teflon–tube was connecting the two chambers. The sample was pumped with a syringe 

pump from the reactor chamber, which was held at 37 ◦ C with a heat wire device, into the 

pre–cooled sampling chamber. The average freezing time for 3.9 µl PBS–buffer was 3.2 s. 

This freezing time is fast enough to freeze, for example, extracellular metabolites in a su- 

pernatant, since the limiting step is the separation of the cells from the supernatant. For 

the inactivation of the cell metabolism, to measure intracellular metabolites, the freezing 

time is still too long, because cell metabolism quenching times of less than one second are 

desirable. These fast quenching times are needed, since a typical metabolite half–life is in 

63

64 7 Concluding Remarks 


PDMS membrane 

Top 

cover 

Cell culture Heat wire device 

Temperature sensor 




Sticky 

tape 

Figure 7.1: Final setup of the bio–microreactor 

Syringe pump 

the order of a second or less [41]. To achieve such fast freezing times in our bio–microreactor 

setup, a better insulation and a new design for the sampling chamber is needed. In the 

present sampling chamber the volume to diameter ratio was limited by the thickness of two 

PMMA plates and the space which was needed for the interconnections. In a new design a 

larger diameter, with less height but the same sampling volume, would be advantageous to 

decrease the freezing time. One possible design could be a cavity for the Peltier element in 

the bottom plate of the sampling chamber, so that the bottom of the chamber is shifted to 

the center of the plate, whereas the height of the chamber is reduced and thus the diameter 

can be increased (Fig. 7.2). 

The next improvement step, for the bio–microreactor, could be a further development to 

a bio–microfermentor. For this goal, primarily, the sterility problem mentioned in Chapter 

6 has to be solved. One solution could be to fill the reactor with medium and close the 

reactor. Subsequently the reactor could be placed for 2 h at 80 ◦ C into an oven. Before 

connecting it again to the sampling chamber, the medium in the reactor chamber can be 

inoculated through the PVC–tube interconnection. This solution is not perfectly sterile, 

because of the too low temperature, thus, in addition, it would be sensible to use a cell 

strain, which can grow in the presence of an antibiotic in the medium. In addition, a mixing 

system has to be integrated to obtain a better aeration. The integration of a magnetic 

stirrer into a small-scale cultivation system was recently published [48]. Also, it would be 

useful to integrate optical oxygen, pH and OD sensors. The integration of these sensors into 

a small-scale cultivation system was already demonstrated [36]. To integrate these sensor 

optical technology, it would be advantageous to use the electrical resistant heat foil device 

instead of the electrical resistant heat wire device, because the heat foil is transparent. Thus, 

further investigations why the heat foil burned several times in our experiments would be 

sensible. If a heat wire will be used, the device has to be created in a way that some regions

7 Concluding Remarks 65 

Present sampling chamber Improved sampling chamber 



Figure 7.2: Possible improvement of the sampling chamber to obtain shorter freezing times 

are free of heat wire, to place the optical sensors there. But this might lead to a non optimal 

heating. 

As mentioned above one application for the bio–microreactor could be the freezing of su- 

pernatant to quench the degradation process of extracellular metabolites. But to obtain 

supernatant, the cell culture has to be filtered. But the integration of a filter into a mi- 

crosystem is nontrivial. The main problem of the commercial available filters is the high 

dead volume. PMMA filters have almost no dead volume, but the research on them is not 

yet sophisticated to produce that small pore size which is needed for the filtration of E. coli 

cells. 

Another imaginable application is the already mentioned cell metabolism inactivation to 

measure intracellular metabolites. Depending on the achievable freezing times of the cell cul- 

ture and the membrane integrity, intracellular metabolite measurements can be conducted. 

But another critical step for measuring the metabolite levels is the conversion of the common 

metabolite extraction methods to the small scale. typically, at the large scale, the prepara- 

tion for extracting metabolites starts with spraying the cell culture directly in −40 ◦ C cold 

60 % methanol solution [41]. To translate this step to small scale, in the present configura- 

tion of the sampling device, a frozen block of cell culture would have to be suspended in the 

above mentioned methanol solution. Whether this frozen block of cell culture leads to the 

same extraction results, will require further experiments. 

In principle, several sampling chambers could be connected to the reactor chamber, which 

would allow to develop a fully automated sampling system, with which the samples are 

frozen until they are used for further measurements. Such a fully automated sampling 

system provides a convenient possibility to sample during a fermentation process without 

user interaction.

Appendix 

Freezing experiments with PBS–buffer concerning the bio–microsystem design 

Experiments with E. coli cells 

Volume [µl] Freezing time [s] 

Design II (PBS–buffer) 20 40 

20 45 

20 50 

20 45 

20 50 

20 50 

20 49 

20 50 

Design III (PBS–buffer) 20 52 

20 115 

20 174 

Design IV (PBS–buffer) 20 90 

20 65 

20 60 

Volume [µl] Freezing time [s] Viability[%] 

Design II (cells, 10 3 cfu/ml) 20 30 86.74 

20 40 58.08 

20 45 91.38 

20 68 79.38 

67

68 Appendix 

Experiments with PBS–buffer concerning the volume (non checked pipettes) 

Volume Freezing Freezing Freezing Freezing 

PBS–buffer [µl] time [s] time [s] time [s] time [s] 

2.5 13 12 16 14 

5 16 16 13 15 

10 22 25 30 33 

15 34 32 20 29 

20 34 30 35 40 

Experiments with LB–medium concerning the volume (non checked pipettes) 

Volume Freezing Freezing Freezing Freezing 

LB–medium [µl] time [s] time [s] time [s] time [s] 

2.5 19 18 18 18 

5 27 26 17 23 

10 19 21 22 20 

15 29 29 33 30 

20 32 37 30 33 

Experiments with PBS–buffer concerning the volume (checked pipettes) 

Volume Freezing Freezing Freezing Freezing Freezing 

PBS–buffer [µl] time [s] time [s] time [s] time [s] time [s] 

2.5 17 15 119 20 12 

5 16 24 19 17 22 

10 33 29 21 19 26 

15 23 33 32 40 29 

20 30 33 41 34 29 

Experiments with LB–medium concerning the volume (checked pipettes) 

Volume Freezing Freezing Freezing Freezing Freezing 

LB–medium [µl] time [s] time [s] time [s] time [s] time [s] 

2.5 13 12 13 12 17 

5 17 16 14 15 16 

10 20 18 21 27 25 

15 28 31 29 28 24 

20 36 32 24 38 29

Experiments with 20 µl PBS–buffer concerning the temperature 

Appendix 69 

Time[s] Temperature [ ◦ C] Temperature [ ◦ C] Temperature [ ◦ C] 

0 23.1 22.0 21.7 

10 16.5 15.6 14.0 

20 9.7 9.2 8.3 

30 6.4 4.9 5.7 

40 5.5 3.0 5.5 

50 4.7 4.5 4.6 

60 3.9 4.1 3.7 

70 3.0 3.8 2.7 

80 2.1 3.1 1.5 

90 0.5 2.4 -0.9 

100 -3.2 1.6 -3.9 

110 -6.5 0.3 -5.2 

120 -7.9 -0.2 -6.0 

130 -8.5 -5.4 -6.3 

140 -8.9 -7.0 -6.8 

150 -9.2 -7.6 -7.2 

160 -9.5 -7.9 -7.4 

170 -9.7 -8.2 -7.7 

180 -10.0 -8.4 -7.4 

190 -10.1 -8.6 -8.2 

200 -10.3 -8.8 -8.3 

210 -10.5 -9.0 -8.5 

220 -10.7 -9.0 -8.7 

230 -10.8 -9.1 -8.9 

240 -10.9 -9.2 -9.1

List of Figures 

3.1 Structure of PMMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 

3.2 Profile of a channel made by an infrared laser system . . . . . . . . . . . . . 19 

3.3 A laser micromaching system . . . . . . . . . . . . . . . . . . . . . . . . . . 20 

3.4 Structure of cyclo–olefin copolymers . . . . . . . . . . . . . . . . . . . . . . . 21 

3.5 Profile of a channel made by a CNC milling machine . . . . . . . . . . . . . 22 

4.1 Illustration of the physical structure of a conventional Peltier effect refrigerator 25 

5.1 Tested designs of the freezing chambers consisting of a laser machined PMMA 

plate and different bottoms . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 

5.2 WinMarkPro drawings of the freezing chambers . . . . . . . . . . . . . . . . 29 

5.3 Experimental setup for freezing a sample using Peltier cooling . . . . . . . . 32 

5.4 Leaking of the freezing chamber . . . . . . . . . . . . . . . . . . . . . . . . . 36 

5.5 Results of freezing PBS–buffer with Peltier cooling in different designs . . . . 37 

5.6 Viability results of freezing E. coli cells with Peltier cooling in freezing cham- 

ber II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 

5.7 Averages of the freezing times against the different volumes of PBS–buffer 

and LB–medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 

5.8 Averages of the freezing times against the different volumes of PBS–buffer 

and LB–medium from the reiterated experiment with a well calibrated pipette 40 

5.9 Temperatures of 20 µl PBS–buffer during the freezing using a Peltier element 40 

6.1 Setup of the bio–microreactor in two different conditions . . . . . . . . . . . 42 

6.2 O–ring interconnection in a channel . . . . . . . . . . . . . . . . . . . . . . . 44 

6.3 WinMarkPro drawing of the mold for the O–rings . . . . . . . . . . . . . . . 44 

6.4 Longitudinal section of the mold for O–rings . . . . . . . . . . . . . . . . . . 44 

6.5 Cross section of the bores for the PVC–tube interconnections . . . . . . . . . 48 

6.6 First reactor chamber setup . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 

6.7 Second reactor chamber setup . . . . . . . . . . . . . . . . . . . . . . . . . . 52 

71

72 List of Figures 

6.8 Heat wire device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 

6.9 The sampling chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 

6.10 Electronic circuit to interface USB LabJack U12 data acquisition and control 

card with the heater and thermistor . . . . . . . . . . . . . . . . . . . . . . . 57 

6.11 Photograph of the bio–microreactorr . . . . . . . . . . . . . . . . . . . . . . 60 

6.12 Average of the freezing time against the different volumes of the sampling 

chambers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 

7.1 Final setup of the bio–microreactor . . . . . . . . . . . . . . . . . . . . . . . 64 

7.2 Possible improvement of the sampling chamber . . . . . . . . . . . . . . . . . 65

List of Tables 

5.1 CO2–laser settings for design I to IV and for the top cover . . . . . . . . . . 30 

6.1 Three different CO2–laser settings for the O–ring mold . . . . . . . . . . . . 46 

6.2 Three changes in the CO2–laser settings for the O–ring mold . . . . . . . . . 47 

6.3 CO2–laser settings for the reactor chamber . . . . . . . . . . . . . . . . . . . 50 

6.4 New CO2–laser settings for the reactor chamber . . . . . . . . . . . . . . . . 51 

6.5 CO2–laser settings for the three sampling chambers . . . . . . . . . . . . . . 55 

6.6 Measured data for freezing PBS–buffer samples . . . . . . . . . . . . . . . . 61 

73

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