Functionalized graphene sensors for real time monitoring fermentation processes Chinnathambi, Selvaraj
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Functionalized graphene sensors
for real time monitoring
fermentation processes
Electrochemical and chemiresistive sensors
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the
Rector Magnificus Prof. C. Wijmenga and in accordance with
the decision by the College of Deans. This thesis will be defended in public on Thursday 30 January 2020 at 12.45 hours
by
Selvaraj Chinnathambi
born on 6 April 1987 in Sattur, India
Prof. Y. Pei
Assessment Committee
Prof. F. Picchioni Prof. B.J. Kooi Prof. H.J. Wortche
Chapter 1: Introduction
1.1 Introduction……….…………. 1
1.2 Bacterial fermentation process……….………… 3
1.2.1. Downscaling bioreactors……….………... 5
1.2.2. Microtiter plate mini reactors...……….……… 6
1.3. Chemiresistor based sensors………... 7
1.4. Graphene………. 9
1.5. contents of thesis………... 12
1.6. References………... 13
Chapter 2: Overview of miniaturised sensor for application in micro
bioreactors
2.1. Introduction………..…….… 232.2. Fundamentals of sensing principles………..…… 24
2.2.1. Electrochemical sensors... 24
2.2.2. Optical sensors... 28
2.2.3. Chemiresistive sensors... 30
2.3. Overview of miniaturised pH and dissolved oxygen sensor………... 31
2.3.1. Electrochemical pH sensor... 31
2.3.2. Electrochemical dissolved oxygen sensor...………....……….….…. 38
2.3.3. Optical pH sensor... 24
2.3.4. Optical DO Sensor... 46
2.3.5. Biomass sensor………..……. 50
2.4. Sensors for microtiter plate mini bioreactors... 53
2.5. References………... 58
Chapter 3: Polyaniline functionalized ERGO chemiresistive sensor for real
time monitoring pH during Lactocauccous Lactis fermentation
3.1. Introduction………..…………. 753.2. Experimental details………..………… 77
3.2.1. Graphite oxide preparation………..………… 77
3.2.2. Electrochemical reduction of Graphene oxide………..….. 77
3.3.2. Potentiometric sensing of ERGO-PA……….…… 83
3.3.3. Chemiresistive sensing of ERGO-PA………... 87
3.4. Conclusion………..….. 94
3.5. References……… 95
Chapter 4: Fabrication of hydrothermally reduced graphene oxide electrode
for potentiometric and chemiresistive pH measurements
4.1. Introduction……….…….1004.2. Experimental details……….….. 102
4.2.1. Material preparation……….…. 102
4.2.2. Electrochemical pH sensing……….…. 102
4.3. Results and discussion……….…... 103
4.3.1. Material characterization………...…... 103
4.3.2. Potentiometric pH sensing of HRGO………..………….. 105
4.3.3. Cyclic voltammetry of pH dependent HRGO……….……….. 110
4.3.4. Chemiresistive sensing of HRGO……….………… 115
4.4. Conclusion……….. 116
4.5. References ……….. 117
chapter 5: Nitrogen and Boron doped reduced graphene oxide chemiresistive
dissolved oxygen sensor: A new approach towards dissolved
oxygen sensing
5.1. Introduction………...…….. 1235.2. Experimental details………...……. 125
5.2.1. Material preparation………..…… 125
5.2.2. Electrode preparation………..……. 126
5.3. Results and discussion……….………….……….. 127
5.3.1. synthesis and characterization of N-B-HRGO……….….……… 127
5.3.2. Amperometric sensing of N-B-HRGO……….……… 130
5.3.3. Chemiresistive sensing of N-B-HRGO……….………… 136
5.4. Conclusion……….………. 144
sensor for online monitoring fermentation process in 3D printed
miniaturised reactor
6.1. Introduction……….… 154
6.2. 3D printing……….……. 156
6.3. Experiment details……….…….. 159
6.3.1. Miniaturised reactor design ……….. 159
6.3.2. Sensor fabrication………. 161
6.3.3. Bacterial culture preparation for sensor measurements in 3D printed reactors… 162 6.3.4. Calibration of the sensors………...163
6.4. Results and discussion………...………. 165
6.4.1. Bacterial growth experiment in 3D printed reactor……….. 165
6.4.2. Real time measurement of pH, DO and Biomass during fermentation process... 166
6.4.3. Wireless sensor network for data readout……….… 170
6.5. Conclusion……….. 171
6.6. References……….. 172
Chapter 7: Summary and future direction
Samenvatting
List of Publications
Acknowledgements
Curriculum vitae
1.1. Introduction
Fermentation is an important biological process that plays a vital role in several industrial applications, for example in the food industry and beverage production. The fermentation processes is usually performed batch-wise in large bioreactors. Bacteria, yeast or fungi grow in a liquid culture medium containing a carbon, nitrogen, and phosphorus source and some other macro- and micro-nutrients depending on the nutritional requirements of the organism. In addition to that, most biological processes in bioreactors are monitored and controlled using one or multiple sensors. Most frequently, sensors for pH, dissolved oxygen, redox potential, and temperature are used for general fermentation process monitoring. If there is a need to optimize a particular fermentation process, ideally this would be performed in the same reactors as the reactors that will be used in the final production process. However, large volume of culture media, additional chemicals, and energy are necessary to maintain optimal process parameters like pH, dissolved oxygen, and temperature are required. In this regard, downscaling of the reactor size is crucial for efficient and economical determination of the effect of variations in process parameters, media composition and organisms. Small-sized fermentors can be the basis for the development of a high throughput screening platform for optimal growth and production conditions. However, extensive validation need to be performed to ensure that the optimal fermentation conditions are the same optimal conditions in large scale reactors. Another challenge with mini-reactors is the necessity of small-sized sensors to monitor and control the process parameters. For an efficient and reasonable priced high throughput screening system, the sensors should be cheap, easy to maintain or replace, and are readout with simple electronics that can be easily multiplexed to avoid the use of expensive multichannel analog-to-digital converters. The system should be simple to build with as little complexity as possible. For instance every reactor contains at least a pH sensor, dissolved oxygen sensor, temperature sensor, a heating element, and some kind of biomass
sensor. With one ground and 5 signals, 6 electrical connections per fermentor are needed. In a 96-reactor system this is 6 x 96= 564 electrical connections. The system becomes even more complicated if the liquid and gas connections are also taken into account. The reactors contain a feed-in, effluent out, base-in, acid-in, gas-in, and gas out resulting in 6 x 96 = 564 small tubes in and out of the reactor. So, more than 1000 connections to the screening platform need to be established. Wireless sensors (Bluetooth, Near Field Connections (NFC)), and/or Wi-Fi for the electrical signals reduces these complexities. Though it increases the prize of the sensor, the wireless sensing system creates the possibility to develop smart process control systems.
For pH measurement glass electrode based electrochemical sensors are commonly used. It is very difficult to miniaturize the glass electrode due to its fragility of thin glass and reference electrode system. Because of this problem, other miniaturized pH sensors based on ISFET and fluorescence are used in miniaturized bioreactors. The ISFET and optical pH sensors also have some disadvantages. Long term stability of ISFET pH sensors is an issue due to drifting of the output signal and fluorescence-based optical pH sensors have a limited sensing range. Even though limitations exist, optical pH sensors are used in microtiter plate based mini bioreactors (Matrix-24, BioLector). In this thesis, we developed chemiresistive sensors for monitoring the pH and dissolved oxygen. We show that the sensors can be applied in an integrated sensing platform for microtiter plate based mini bioreactors potentially to be expanded into a high throughput fermentation screening platform.
1.2. Bacterial fermentation Process
Fermentation is a complex biological process that is dependent on the environment and also changes the environment. Fermentation is performed by the microorganism that belongs to all three kingdoms, Bacteria, Archaea and Eukarya. Fermentation is defined as the process in
which the microorganisms generate their own electron acceptors if an external electron acceptor is not available or cannot be used by the microorganisms. The electron acceptors are reduced by the protons and electrons derived from the electron donor. Usually, the electron donor is the carbon source but hydrogen can also be used as the electron donor. The products are excreted e.g., short chain volatile fatty acids (acetate, propionate, butyrate), lactate, ethanol, butanol or methane. The excretion of these fermentation products led to a large number of food, feed, and energy applications by adding value, like taste, structure, and energy conversion to the source material. Nowadays, fermentations are an important application area in the industry. Process control during the fermentation process is critical to obtain optimized growth and production conditions.
Figure 1: Illustration of process parameters affecting the fermentation process
There are several process parameters that influences the bacterial fermentation. Among them, pH, dissolved oxygen, biomass, redox potential, and temperature play a crucial role in determining the quality of the fermentation process. For lab and industrial scale bioreactors,
Bacterial Fermentation pH O2 2 (DO) Temperature CO2 Biomass concentration Redox potential
conventional electrochemical (pH, O2, and redox), optical (pH, O2, and biomass), and
capacitance sensors (biomass) are normally used to the satisfaction of the process engineers. However, when bioreactor research follows the current trend of miniaturization and parallel processing, the traditional sensors are too big to fit into the small reactors and too expensive to apply in high throughput screening systems. Besides the online sensors for continuous process monitoring, there are several analytical methods used to monitor and control the fermentation process. In the past, classical analytical techniques like liquid chromatography, gas chromatography and mass spectroscopy and other analytical devices were used off-line. The progress in automatization and robotics enable the researchers to integrate the analytical equipment into online monitoring tools of fermentation processes.
1.2.1. Downscaling of Bioreactors
Small scale bioreactors are excellent tools for the screening of optimal fermentation conditions. They also might be useful in the selection of suitable microorganisms for the development of a new fermentation process. They might even serve as production reactors in continuous flow systems analogues to continuous-flow parallel microreactors in the chemical industry.
Miniaturization of cultivation systems has several effects on the physicochemical property of microorganisms and the operation of bioreactors [1]. Though the dimensional change of suspension of culture in a miniaturised reactor, compared to standard conical flask, has minimum effect on the physiology of the microorganism, there are other effects that dominates the culture in miniaturised reactors. A higher specific oxygen transfer rates (OTRs) is obtained in miniaturised culture systems due to high gas-liquid exchange area to the volume of the bulk liquid. Another important factor dominating the miniaturization effect is the surface tension of the culture medium. Microtiter plates have been successfully used as
miniaturised bioreactors and they become one of the important devices in the process of downscaling bioreactors because they are cheap, widely available in a variety of formats and construction material is sterile and compatible with a growing number of downstream analytical methods (Microtiterplate (MTP) readers, auto samplers, robotic liquid handling equipment, centrifuges, DNA extraction methods, etc.). The microtiter plates are increasingly being used instead of standard conical flask as a high throughput miniaturised reactor.
1.2.2. Microtiter plate mini bioreactors:
Microtiter plates have been used extensively as a high throughput screening platform for strain, gene library selection, or simple growth condition optimization as an alternative to multiple shaken flasks for screening large numbers of fermentative/bioconversion processes. However, screening experiments with concomitated monitoring and control of fermentation parameters (pH, DO, and biomass) in microtiterplates or multiple individual microreactors are limited [1-3]. Some advantages of using microtiterplates as a screening platform feature simultaneous investigation of different medium composition, effect of physiological conditions like pH and oxygen transfer rate, mixing and diffusion of reactants in the media. In addition to that, the standard microtiter plate is compatible with existing automated system and uses a small footprint [4-6]. Some newly designed mini bioreactors also are equipped with small magnetic stirring bars or gas-induced impellers to improve mass transfer to the culture. The growth of Escherichia coli in such mini-reactors was compared and it was found to be equivalent to that in larger scale (up to 3 L) bioreactors [7-8]. Pharmaceutical companies and research laboratories are looking closely at small-scale bioreactors to meet the needs of higher throughput controlled cell cultivation [5, 9-12]
Figure 2: Image of a standard 96-well microtiter plate
To use microtiter plate as a high throughput screening platform with online monitoring and control of process parameters, the miniaturised pH, dissolved oxygen, biomass, and temperature sensors needs to be implemented within the microtiter plate. The real time online measurements of pH, dissolved oxygen and biomass provide overall quantitative measurement of medium composition and culture growth. The integrated sensor platform for microtiter plate is investigated in the past using conventional electrochemical sensing techniques, for example, amperometric dissolved oxygen sensor and ISFET pH sensor [13]. These conventional sensing methods use reference electrodes and need special equipment for signal readout. It makes this integrated sensing platform not viable for commercial MTP bio reactors.
1.3. Chemiresistor based sensors
Chemiresistor is a material that changes the resistance/conductivity to a large extent when the chemical environment changes. The chemiresistor consists of two electrodes separated by a distance. The electrodes are connected by chemiresistor material. The resistance or conductivity of the material is measured by applying a constant voltage between the two
electrodes and record the resulting current. The sensor response of the chemiresistor is obtained by measuring the change of the resistance or current of the chemiresistor material upon interaction with the analytes. The sensing mechanism of chemiresistor sensor can be explained by three phenomena which can occur synergistically or independently. The resistance or conductivity change of the chemiresistor device can occur due to a change of doping level, modulation of the Schottky barrier, or, modulation of the junction resistance between particles. When analytes interact with chemiresistor material, the doping level is changed due to change in the concentration of the charge carriers. The effect of doping depends on the nature of the semiconductor material.
Figure 3: Schematic representation of a chemiresistor device
The electron donating analytes, for example NH3 injects an electron and the resistance of the
p-type semiconductor increases while the resistance of the n-type material decreases. Similarly, the resistance of the p-type material decreases and n-type material increases if the analytes are electron withdrawing groups. When analytes interact with the surface, the work function of material changes which results in the modification of the Schottky barrier between
Au electrode
Reduced Graphene oxide
Polyaniline
Hole proton
H+
the electrode-material contact. When analytes adsorb at the grain boundaries or particle junction the hopping distance increases due to an increase in the distance between the grain boundaries. A good chemiresistive sensor has features like fast response time, low drift, fast recovery, high sensitivity, and long term stability.
Several materials are used as chemiresistor, mainly, for gas sensing applications. Carbon nanotubes (CNT) [14-16], graphene [16-17], metal nanoparticles [18-25] and conducting polymers [26-28] are widely studied as chemiresistive material in gas sensing applications. Recently, a CNT-conducting polymer composite and a gold nanoparticle film are used as chemiresistor material in sensors for applications in aqueous solution [29-31]. Only a few chemiresistor have been developed to detect analytes in aqueous solution. Hence, it is highly challenging and also important to develop chemiresistor devices for the detection of analytes dissolved in aqueous solutions.
One of the significant advantages of Graphene is that its properties can be tuned by functionalization, giving modified graphene a considerable potential in sensing applications. Responsive molecules can be attached covalently or non-covalently onto the graphene surface using (electro-)chemical methods. In addition to these properties, the chemical stability of graphene is advantageous in chemically harsh environments.
1.4. Graphene
Graphene is a two-dimensional single atom layer that consists of an sp2-bonded carbon
network. Graphene was discovered by A. K. Geim and K.S. Novesolo by mechanical exfoliation of graphite using the famous scotch tape method [32-33]. Graphene has excellent properties like high electron mobility, thermal conductivity and high mechanical strength [34]. Mechanical exfoliation of graphene is an easy way to prepare high quality graphene, however, the production of large quantities for commercial application is cumbersome .
Hence, there are several methods of graphene preparation in development. Chemical vapor deposition, reduction of graphene oxide, electrochemical exfoliation of graphite, thermal decomposition in the presence of volatile compounds are some of the methods that are explored for graphene synthesis at a larger scale . Among them, production of graphene by chemical graphene oxide reduction (RGO) is a promising method to produce graphene on a larger scale.
Graphene is produced chemically by reducing graphene oxide. Graphite oxide is a precursor for RGO preparation. Hummers and Offeman developed a method for graphite oxidation [35]. They used potassium permanganate and concentrated sulfuric acid as oxidizing agents. A similar approach was developed by L. Staudenmaier using a mixture of KClO3 and sulfuric
acid to oxidize graphite [36].
Chemical production of graphene involves oxidation of graphite into graphite oxide, exfoliation of graphite oxide into graphene oxide, and the reduction of graphene oxide to graphene (RGO). There are several ways in which graphene oxide can be reduced to graphene. Graphene oxide can be reduced by various reduction methods among others chemical, thermal, electrochemical and photo catalytic reduction [37-60]. In the chemical approach, reducing agents such as hydrogen sulphide, hydrazine, or sodium borohydride are used under hot alkaline conditions (pH 10 at 90 ºC) [44-51]. For thermal reduction, graphene oxide is heated to 200-300 °C for one hour. During this treatment, the functional groups are removed by thermal decomposition, hence, hazardous gaseous species are liberated [52-54]. Electrochemical reduction of graphene oxide is considered as a much more green and environmental friendly reduction process. Graphene oxide is deposited on a conducting substrate. The reduction occurs around -0.8 V (Ag / AgCl) and the majority of the functional groups are removed by this method [55-57]. UV-assisted photocatalytic reduction is also used to reduce graphene oxide. In this process, reduction occurred by charge transfer reaction
between TiO2 and graphene oxide. During irradiation, the surface charge of TiO2 is increased
which is used for reduction of functional groups that are present on the graphene oxide [58]. RGO is also prepared by a hydrothermal reduction method. A GO dispersion is placed in a Teflon lined autoclave and heated upto 120-180 °C. In this process super-heated water molecules are involved in the reduction of GO [59-60].
Reduced graphene oxide contains defective sites due to the removal of oxygen containing functional groups and the reduction of GO is not always complete. This means that there are always some oxygen functional groups left on the RGO. The defects and left-over oxygen functional groups renders RGO as a high reactive material for further functionalization to make it useful in sensing applications [66-66]. Several, RGO based sensing devices are fabricated as gas and molecular sensors. RGO can be deposited as thin film, flakes, or rippon form on a e.g., interdigitated gold electrode. Chemiresistive sensing applications have been reported for NH3, NO2, O2 and volatile organic compounds but not for aqueous applications
1.5. Contents of the thesis
Chapter 1 introduces the objective of this thesis and explains the importance of downscaling
the bioreactor and the role of a sensor platform in it. As the graphene is used as a sensor material, its properties and the available methods of synthesis also are introduced in this chapter.
Chapter 2 describes the process parameters affecting the bacterial fermentation process and
the fundamentals of the sensor used for process monitoring. An overview of micro sensors for the miniaturised micro titer plate bioreactor is presented in this chapter.
In chapter 3, the fabrication of a polyaniline functionalized electrochemically reduced graphene oxide pH sensor is reported. Potentiometric and chemiresistive sensing of this material is explored and the results are discussed. The feasibility of this pH sensor to monitor the pH during the bacterial fermentation process is shown.
In chapter 4, the pH sensing property of hydrothermally reduced graphene oxide is investigated. Potentiometric and chemiresistive pH sensing of hydrothermally reduced graphene oxide is explored and results are discussed. Cyclic voltammetry is used to characterize the nature of functional groups present on the reduced graphene oxide.
In chapter 5, a Chemiresistive dissolved oxygen sensor based on nitrogen and boron doped hydrothermally reduced graphene oxide is explored as a new approach towards dissolved oxygen sensing. Material preparation, characterization, amperometric and chemiresistive sensor properties results are presented and discussed in detail. Further, the sensor is tested in a bacterial fermentation process and the results are discussed in this chapter.
Chapter 6 presents an integrated sensing platform for miniaturised microtiterplates based
bioreactor is explored and optical biomass sensor is developed to measure the optical density of the bacterial growth. The fabrication of integrated sensor lid containing chemiresistive pH, dissolved oxgyen and the fabrication of optical biomass sensor results are discussed. The miniaturized reactor with sensor housing lid is fabricated using 3D printing technology. The growth of bacteria in the 3D printed miniaturised bio reactor is investigated and the results are discussed in this section.
In Chapter 7, the complete thesis work is summarized and the future direction to improve the sensor properties are discussed.
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Overview of miniaturised sensors for application in
microbioreactors
2.1. Introduction
Development of fermentation processes in bioreactors usually requires many experiments because many conditions influence the outcome of a fermentation. If only a small number of reactors is available, the development takes a long time. If multiple reactors are available, laboratory space, investment and operating costs, and handling of the reactors by the researchers become more of a challenge. A possible solution could be downscaling of the reactors to make efficient use of space, labor, and investments. To ensure compatibility with commercially available analytical equipment and molecular/biochemical kits, the format of a microtiter plate (MTP) is preferred. Various commercially available MTPs exists with different heights and number of wells. The number of wells ranges from 6 to 3456, with volumes ranging from 17 ml to 2.7 µl.
Fermentation processes are highly sensitive to many process parameters that need to be measured online and controlled to specific set points. Among the process parameters, pH, dissolved oxygen (DO), biomass, and temperature are the main parameters that are of general importance. Hence the bioreactors should always be equipped with sensors to monitor the process parameters. In laboratory (bench-top) reactors with volumes from l to 10 liters, electrochemical sensors are used to monitor the pH and dissolved oxygen. For pH, glass-based electrodes are used, and Clark-type electrodes are used for DO measurements. These electrochemical sensors are difficult or impossible to use in small, MTP-based, microreactors because of size constraints.
Ion-Sensitive Field-Effect Transistor based electrodes (ISFET) have the potential to be used in microreactors because they can become very small. Commercially available ISFET pH sensors are successfully integrated with micro reactor arrays for process monitoring. The drift in voltage during the measurement was reported to be one of the problems associated with ISFET pH sensors. However, the drift is stable after two hours and is reproducible [1-5].
Optical sensors are another category of sensors that are available to detect pH and DO in microreactors. Optical pH and DO sensors have been successfully used and integrated with micro reactor arrays. The useful pH detection range is narrow for the optical pH sensor, and it is challenging to measure pH in processes carried out at pH <5 [6].
2.2. Fundamentals of sensing principles
Though there are many sensing methods available, only the sensing methods relevant for fermentation processes are considered here. As mentioned earlier, several parameters are important for fermentation processes. However, only the sensors available for crucial parameters like pH, DO, biomass, and temperature is mentioned in this chapter.
In this section, the fundamentals of an electrochemical sensors, optical sensors, impedimetric sensors, and chemiresistive sensors are discussed briefly.
2.2.1. Electrochemical sensors
There are two ways of how a signal is obtained from an electrochemical sensor, one is potentiometric, and another one is amperometric. In potentiometry, the potential of the working electrode is measured with respect to the concentration of an analyte when a fixed current is applied to the electrode. In amperometry, the current generated during the electrochemical reaction at the working electrode was measured at a fixed potential against the reference electrode. A typical electrochemical system is shown in Fig. 1. An electrochemical system
consists of three electrodes: the working electrode, the reference electrode, and the counter electrode. The potential applied to the working electrode is always referred to as a common reference electrode (Hg / HgCl2, Ag/AgCl, reversible hydrogen electrode) [7-8].
Figure 2.1: Schematic representation of an electrochemical cell. WE: working electrode; CE:
counter electrode; RE: reference electrode.
2.2.1.1. The potentiometric method
In potentiometric methods, the equilibrium potential of the working electrode is measured. The potentiometric sensor is the basis of many electrochemical pH sensors. With this technique, the equilibrium potential of the ion selective electrode with respect to the Ag/AgCl reference electrode is measured. The equilibrium potential of a single electrode cannot be measured directly, and hence it is always connected with the non-polarizable electrode (reference electrode) that has a constant potential. The standard saturated calomel electrode (Hg / HgCl2, SCE), and Ag / AgCl electrode are mostly used as reference electrodes. Traditionally,
the indicator electrode, whose equilibrium potential needs to be measured, and the reference electrode are placed in different solutions that are separated by a salt bridge [7,8].
+
-WE
RECE
Voltage is monitored Current is measuredThe equilibrium potential of the electrode varies with the activity of the ions that are present in the solution. The potential difference between the ion selective electrode and reference
electrode occurs due to change in the activity of the ions according to the Nernst equation (Eqn.1).
Equation 1
Where, Eo is the standard electrode potential, E is an equilibrium potential, a
o and ac is the
activity of oxidized and reduced species.
2.2.1.2. The amperometric method
In the amperometric method, the electrical current that arises from the electrochemical reaction of an electroactive species at the electrode surface is measured. The externally applied potential on the electrode with respect to the reference electrode drives the electrochemical reaction. The value of the current during the electrochemical reaction is directly proportional to the concentration of the electroactive species. The diffusion of the reactants from the bulk solution controls the concentration of the reactants at the electrode surface. The resulting current follows
the Cottrell equation (Eqn 2.) [9,10]. 𝐼 =𝑛𝐹𝐴𝐶𝑜𝐷1 2⁄
𝜋1 2⁄ 𝑡1 2⁄ Equation 2
Where I is the current, n is the number of electrons, A is the area of the electrode, F is the Faraday constant, D is the diffusion coefficient, Co is the concentration of the electroactive
species in the bulk of the electrolyte, and t is time. Eqn. 2 is only applicable to planar electrodes. During amperometric detection, the electroactive species is reduced at the electrode surface, and a diffusion layer is formed due to the concentration gradient. The diffusion layer, and therefore the signal of the electrode, is flow dependent. An ultra-microelectrode can be used to mitigate the problem of the flow-dependent current response. In this case, the oxygen
concentration gradient is formed in a few milliseconds, and the diffusion of oxygen is so high that convection no longer affects the reduction process. For a circular-disc ultra-microelectrode, the Cottrell equation (Eqn. 3) becomes
Equation 3 Where I is the current, n is the number of electrons, A is the area of the electrode, F is the Faraday constant, D is the diffusion coefficient, Co is the concentration of the electroactive
species in the bulk of the electrolyte, and r is the radius of the disc. Eqn. 3 indicates that the current response no longer depends on time [9-11]. For some microscale applications, an array of microelectrodes has been used to increase the sensitivity. When an array of electrodes is used, the electrodes should be positioned in such a way that the diffusion path of the electroactive species from the bulk solution to the individual electrodes is not merged [12-13]. Recessed electrodes are used to reduce the spacing between the electrodes. Recessing is also used in conventional Clark polarographic electrodes to avoid the usage of a polymer membrane. The recessed electrodes reduce the mass transport of electroactive species to the electrode surface, which depends on the height of the recess. The resulting current from recessed electrodes follows Eqn. 4 [14].
Equation 4
Where I is the current, n is the number of electrons, A is the area of the electrode, F is the Faraday constant, D is the diffusion coefficient, Co is the concentration of the electroactive
species in the bulk of the electrolyte, r is the radius of the disc, and h is the height of the recess.
2.2.1.3 Ion-Sensitive Field-Effect Transistors (ISFET)
The construction of an ISFET is similar to a Metal-Oxide-Semiconductor Field-Effect Transistor (MOSFET). In a MOSFET a metal gate is deposited on top of an insulator (metal oxide) overlaying a semiconductor in which two metal connectors, a source, and a drain are
deposited. In the case of an ISFET, the potential of the gate is separated in the form of a reference electrode inserted in an aqueous solution which is in contact with the gate oxide. When an ISFET is exposed to an aqueous solution, the surface of the gate oxide is hydrated. The hydration of the gate oxide changes the concentration of the surface charge, which results in a different threshold voltage of the device. Hydration of the gate oxide is based upon the site-binding model [15-17]. To sense the ions of interest, an ion-selective membrane is placed over the gate to allow that only the specific ions are deposited on to the gate oxide [18-23]. A schematic representation of an ISFET pH sensor is shown in Fig.2.
Figure 2.2: A schematic representation of a MOSFET (a), ISFET (b), and an electrical circuit
diagram of an ISFET pH sensor (c).
2.2.2. Optical sensors
Optical sensors are based upon a change in optical properties of indicator molecules dissolved in a solution or embedded in a polymer matrix. Absorption, fluorescent or luminescent properties of indicator molecules can be used to detect the presence of specific molecules and to determine their concentration. In the case of absorption, light is transmitted through a solution containing the indicator(s) and the transmitted light is detected with a light-dependent sensor (Light Dependence Resistor (LDR) or photodiode). The transmitted light has
the same wavelength but has a different intensity as the incident light from the source [24-26]. The molecule of interest may have the right spectral properties to be detected directly, e.g., pigments. Usually, a derivatization reaction with an indicator dye is necessary to use absorbance-based optical sensors.
Optical pH sensors do not measure the pH directly but measure the concentration of an indicator molecule whose optical properties are dependent on the pH [24-25]. In the case of fluorescence, an indicator molecule is excited at a particular wavelength and emits light at a wavelength higher than the wavelength of the excitation light [24, 27-28]. In pH measurements, the emission of the indicator molecule is related to the concentration of protons, hence with the pH. In DO measurements, quenching by oxygen of the emitted fluorescence intensity of an indicator molecule is used as the detection method. In general, optical sensors measure the activity of ionic sensing species (hydronium ion, pH) and for electron-neutral species like O2 and CO2 they
measure the concentration [24].
Optical fiber sensors use two separate optical fibers to pass light to and from the sample, respectively. An LED is connected at the beginning of the fiber to send light to the sample. A photodiode is attached at the end of a second optical fiber to receive the light from the sample. Fiber optics are not disturbed by electronic noise, have no liquid junction problems, and there is no need for separate reference electrodes. However, optical sensors are sensitive to stray light, turbidity, and gas bubbles in the liquid [29].
In fluorescent sensing, different measurement methods are used. One is based on the luminescent intensity quenching due to radiation-less energy transfer and lifetime of the luminescence excite state (frequency-time domain) [30-32]. The ratiometric measurements are favored over single wavelength direct fluorescent sensing. The ratiometric method offers advantages like reduced interference, overcome photo bleaching, and photo stability issues of fluorescent dyes. The fluorescent dye molecule used for ratiometric sensing should have
overlapping excited and emitted wavelengths so that a single light source and photodetector can be used for the measurements. For fluorescence-based pH sensors, the frequency domain based dual lifetime referencing method is mostly used [33-35].
2.2.3. Chemiresistive Sensors
A chemiresistive sensor is based upon materials that show differences in the resistance/conductivity when the chemical environment changes. The resistance/conductivity change may occur due to swelling of the material, doping, and/or redox reactions with the analytes (Fig. 1.3). The conductivity of the material is measured by applying a constant voltage to the electrodes and measuring the resulting current. The conductivity of the material either increases or decreases depending upon the analyte interaction and results in a characteristic I-V curve. If the charge is removed from the material, the conductivity moves into the negative direction, and it moves toward the positive direction if electrons are injected into the material. The conductivity depends upon the surface charge of the molecules. The conductivity is related to the concentration of the surface charges.
2.3 Overview of miniaturised pH and dissolved oxygen sensors 2.3.1. Electrochemical pH sensor
The glass pH electrode is one of the most popular potentiometric pH sensor and consists of a pH-sensitive silicon membrane whose potential depends on the proton concentration. The glass pH electrode is the most preferred choice in industrial applications because of the high sensitivity and selectivity. However, the size of the rugged glass electrode and the requirement for frequent calibration does not allow it to use them in miniaturised applications, e.g., microreactors or other microfluidics devices. Instead, ion-selective membrane pH sensors and other electrochemical pH sensors have been used in miniaturised applications. In this section, an overview of electrochemical sensors and their use in microscale applications is given.
2.3.1.1. Polymeric membrane ion-selective electrodes
Conventional ion-selective electrodes (ISEs), e.g., glass electrodes, use a liquid contact for the ion to electron transduction. A solid Ag/AgCl electrode dipped in a chloride-containing electrolyte is used as the transducer for ion to electron transduction. Modern potentiometric ISEs use a solid-state electrode as the transducer that allows the fabrication of miniaturised ISEs. The solid-state transducer is coated between the ion-selective membrane and the conductive electrode. Several electro-active compounds are used as solid contact in ISEs, e.g., conductive polymers, carbon materials, and graphene.
The ion-selective membrane can be a thin film, as in the glass electrode, or an impregnated polymer matrix containing ionophores that react or form complexes with the analytes to give a potentiometric signal. Ionophores can be neutral, positively or negatively charged depending on the analyte of interest [36-40]. Polyvinyl chloride (PVC) is the most used polymer in ISEs, because of the high sensitivity and selectivity. However, poor biocompatibility, too brittle, less flexible are drawbacks of PVC. To make the PVC membrane more flexible, plasticizers are often used. Ion-selective polymeric membranes are a mixture of polymers, plasticizer, ionophores, and lipophilic salts. Lipophilic salts are used to delay the Donnan failure. The Donnan failure occurs due to the partition of opposite charges in the membrane after the attraction of analyte ions into the membrane. Several macrocycles and non-macrocycles based neutral carrier ionophores are developed for alkali and alkaline-earth metal ions based ISE’s [41-48].
The first polymeric membrane pH-sensitive electrode that was developed used mesoxalonitrile p-(octadecyloxy)-m-chlorophenylhydrazone as the charged H+ carrier. This charged ionophore was incorporated into a block copolymer matrix. Similarly, 3-hydroxy-N-dodecylpicolinamide charged H+ carrier was used for a pH electrode with an extended pH range [49-51]. Simon et al developed a pH-sensitive electrode using tri-dodecylamine as a neutral charge carrier [52-57].
This neutral ionophore is incorporated into a PVC membrane and showed excellent sensitivity and selectivity within the pH range of 4.5 to 11.0. Many neutral ionophores (e.g., pyridines, piperazines, morpholines, imidazoles, pyrazoles, anilines, diamines) with different pKa values were tested to extend the working pH range of pH electrodes [57-60].
Ion-selective micro pH sensor
Ion-selective micro-electrodes to measure the pH in physiological applications are fabricated using double-barreled glass micropipettes. One of the barrels is used as the working electrode and is filled with pH-sensitive compounds. The other barrel is used as the reference electrode containing Ag/AgCl. Before filling, the working barrel was made hydrophobic by dipping into a trimethylchlorosilane solution. The working electrode is backfilled with a solution of ions to be measured, e.g., a NaCl solution to measure Na+ ions. The reference
barreled electrode is backfilled with a NaCl / KCl solution. The ion-selective micro pH sensor was used to detect pH changes in brain tissues [61]. The neutral carrier ionophores are mostly used to measure the pH in extra and intracellular applications.
Recently, a concentric ion-selective microelectrode was constructed to overcome the slow response time and high noise to signal ratio due to the electrical resistance of an ion exchange cocktail. Two thin borosilicate capillaries with different diameters were used to make a concentric ion-selective micro-electrode. The concentric ion-selective micro-electrode with fast response time and high sensitivity was used for in vitro extracellular studies. The microelectrode was fabricated, as shown in Fig. 4. The response time was obtained from constantly switching the pH solution from 7.42 to 6.87, and an average time constant of 14.9±1.3 ms with a sensitivity of 61.8±2.6 mV/pH was obtained. The applicability of this microsensor was studied in hippocampal slices of rat brain tissue [62].
Figure 2.3: Double-barreled ion-selective polymer membrane micro-electrode for pH measurement (A),
pH buffer solution dispenser for calibration set up (B), electrode response for H+ (C) and Ca2+ (D) ions
[62].
2.3.1.2. Solid-state metal oxide micro pH sensor
Solid-state metal oxide electrodes are another class of potentiometric sensors that can be miniaturised by microfabrication methods. IrO2 is mostly used as the metal oxide electrode for
pH sensing because of its wide pH range, a high sensitivity, and fast response time in combination with low potential drift. IrO2 can be prepared in many ways, e.g., oxidation of an
Ir microelectrode, anodic electrodeposition, thermal oxidation, or sputtering methods [63]. A solid-state IrO2 micro-electrode array was fabricated on a silicon substrate and used to
analyze tissue trauma during the implantation of a sensing probe. The micro-sized Ir array with an area of 700 µm2 and with a 100 µm spacing was fabricated on a silicon substrate (Fig. 5).
The iridium micro-electrode was electrochemically oxidized to form a multilayer hydrous iridium oxide layer. This IrO2 micro-electrode showed a super Nernstian sensitivity of -90
mV/decade with reduced sensitivity of -85.8 mV/decade after exposing the electrode into a 0.1 M phosphate buffer solution. The micro-electrode showed good selectivity against Na+, K+,
Ca2+, and Mg2+ ions, but it also showed a response in the presence of ascorbic acid. A Nafion
dip-coating was applied to suppress the response towards ascorbic acid [64].
Figure 2.4: IrO2 micro-electrode array on printed circuit board (A), micro-electrode connected to a
circuit board for signal processing (B), automated insertion tool for insertion of the pH probe into tissues (C), and a cortical cup used for pH measurements (D) [64].
2.3.1.3. Ion-sensitive FET (ISFET) pH sensor
ISFET pH sensors are widely used as microscale electrochemical sensors in miniaturised applications. In an ISFET pH sensor, the gate oxide insulator is exposed to ions that are present in the test solution. The threshold voltage of the gate oxide changes when the gate oxide surface is hydrated. The adsorption of proton ions on the gate oxide layer is based on the site-binding model [65]. One of the disadvantages of ISFET pH sensors for long term application is that it shows transient behavior after a few hours of operation, this is called drift. However, the drift behavior is reproducible, and therefore, a correction can be used to overcome the drift problem [66]. There are several gate oxides insulators reported, e.g., SiO2, Si3N4, Al2O3, and Ta2O5
ISFET pH sensor for miniaturised application
The micro ISFET pH sensor is used to monitor the pH in physiological applications and microfluidic devices. In general, the ISFET sensor has a miniaturised FET electrode, but it needs an Ag/AgCl reference electrode to control the gate potential. The attempt has been made to solve the problem of the reference electrode by placing it in a separate compartment bridged with the test solution. However, it is also possible to microfabricate the reference electrode along with the FET. Here an overview of recent research that used integrated ISFET pH sensor for physiological application is mentioned.
An ISFET pH sensor was used in real-time pH measurements of sweat in healthcare monitoring devices. An Al2O3 / SiO2 di-electric layer was used as the pH sensitive membrane.
The di-electric layer was deposited on top of an InGaZnO (30 nm) semiconductor channel that acted as the FET substrate (Fig. 6). The holes were created on the Al2O3 layer for the reference
electrode. Subsequently, the Ag/AgCl reference electrode was printed and examined under an optical microscope. The sensitivity of the device for the pH was 51.2 mV / pH [65].
Figure 2.5: Illustration of ISFET device fabrication (a), Optical image of the reference
electrode integrated in the ISFET device structure (b), and real time pH measurement from sweat (c).
An ISFET pH sensor was integrated within a micro fabricated cell culture platform [66]. The device was used to measure the rate of acidification in an extracellular medium. The pH was used to monitor biofilm formation and bacterial metabolic activities. A Micrococcus luteus biofilm formation was detected in a microfluidic environment using an ISFET pH sensor. The ISFET device was fabricated using a Ta2O5 membrane. The pH sensitivity of 50 mV/pH was
obtained for pH range 4-8. The biofilm formation of M. luteus was detected by monitoring the acidification and alkalization of the culture medium during the growth. It was reported that biofilm formation occurs at the bottom of the microfluidic channel during the alkalization phase [67].
An ISFET pH sensor was also used to measure the bacterial activity of Lactobacillus
acidophilus [68]. The activity of L. acidophilus was studied in different sugar solutions. L. acidophilus produces lactic acid during growth and decreases the pH of the medium. The PDMS
based micro tank was created to reduce the volume of the sample to 1 µl. The micro ISFET pH sensor was fabricated on an n-type silicon substrate. SiO2 and Si3N4 insulators were used as the
pH-sensitive gate surface. Similarly, an ISFET pH sensor was used to monitor the metabolic activity of sugar fermentation during the growth of Lactobacillus curvatus and Lactobacillus
sakei. An ISFET pH microsensor was used to differentiate between L. curvatus and L. sakei
bacterium as both microorganisms have different metabolic activities for ribose fermentation.
2.3.1.4. Carbon fiber microelectrode voltammetric pH sensor
The carbon fiber (CF) micro-electrode is another miniaturized version of the sensor that is used in microscale physiological applications. Fast-scan cyclic voltammetry (FSCV) is often used for in vivo pH measurements. CF micro-electrodes are mainly used for real-time monitoring of the pH in brain tissue. CF is an attractive substrate because of its high conductivity, biocompatibility, high mechanical and chemical stability, and the surface can be easily functionalized. In addition to that, the small size of a CF microelectrode has an advantage that
it minimizes tissue damage, has a high spatial and temporal resolution and also can be used for longer time measurements [69].
CF microelectrodes are fabricated by the pulled capillary tube method (Fig. 7). Typically, 50-200 µm carbon fiber is extruded from a capillary tube and acts as the sensor probe. FSCV is used for detection of the pH. A cylindrical CF microelectrode with a length of 50-100 µm was prepared by sealing the CF in a capillary tube or pipette tip as shown in Fig. 7. The pH was detected by keeping the electrode at -0.6 V vs Ag / AgCl reference electrode, and then the electrode was fast scanned at 400 V/s back to 1.4 V in a triangular waveform. After subtracting the background current, differential pH values are obtained, absolute pH values can not be measured using this method [70].
Figure 2.6: Illustration of voltammetric pH sensing using a carbon fiber microelectrode [71]
Modified cylindrical CF is investigated as a reagent-less sensing probe for real time measurement of pH in biological microenvironments. Fast blue RR salt (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chloride) salt), a quinone-containing diazonium derivative, is electrochemically grafted on CF. The 5 µm wide and 200 µm long CF microelectrode was fabricated by aspirating 5 µm CF into a borosilicate capillary tube. FSCV was used for pH measurements. A sensitivity of 39 mV / pH was obtained for a pH range from 6-8.5 in adult hemolymph like (AHL) saline solution. This modified CF microelectrode was used to measure the in vivo pH change in descending neurons (DNs) from the fruit fly
2.3.2. Electrochemical dissolved oxygen sensor 2.3.2.1. Amperometric dissolved oxygen sensor
There are several sensing methods available to detect dissolved oxygen (DO). Among them, electrochemical and optical sensors are preferred for online measurements. The Clark-type polarographic electrodes are amperometric sensors to detect DO. A Clark-type sensor consists of a cathode and an anode. Platinum is used as the cathode and Ag/AgCl is used as the anode. The platinum is separated from the solution by an oxygen permeable membrane. Dissolved oxygen diffuses through the membrane and is reduced at the platinum surface. During the reaction, the silver anode is oxidized and consumed. The electron flow (current) from the silver anode to the platinum cathode is used for the measurement. During the oxygen reduction reaction, oxygen is consumed at the electrode surface, and the concentration of oxygen reaches zero. In an unstirred solution, a concentration gradient is formed near the electrode, which is further extended to the bulk. It is a diffusion or Nernst layer. Hence, the current response of the electrode is controlled by diffusion. In a Clark electrode, a polymer membrane is used to separate the cathode from the solution, and the permeation of oxygen is directly proportional to the concentration of the oxygen in the solution. In an unstirred solution, the presence of the diffusion layer leads to erroneous DO values. This can be avoided by stirring the solution but the stirring rate affects the measurements as well. However, when using microelectrodes the effect of the stirring is minimal [10]. There are two ways DO microelectrodes can be fabricated, one is using pulled glass pipettes, and the second one is using lithographic microfabrication methods. In the next section, an overview of the development of microelectrodes and its physiological applications will be reported.
Microelectrodes for dissolved oxygen
Needle type recessed microelectrodes are mostly used to mitigate the problem of tissue damage, microcirculatory disturbance, and improved spatial resolution [75]. The recessed tip formation
overcomes the flow-dependent current response due to the reduced diffusion layer thickness. In a conventional DO microelectrode, the recessed cathode is prepared in a pulled glass micropipette with a tip of 2-5 µm. Then the pulled micropipette was half-filled with low-melting Bismuth alloy, and then gold was deposited on top of the alloy. The tip of the glass micropipette was recessed using 1 M KCN to dissolve part of the gold (Fig. 8b) [76].
Figure 2.7: Recessed microelectrode fabrication using MEMS (a) and using conventional pulled
capillary tip (b) [75]
The needle type DO micro electrode was fabricated using MEMS technologies [77]. The micro sized glass probes were diced from the wafer. The tip of the glass was etched by dipping in a mixture of HNO3, H2O2, H2O (10:7:33) to form a needle type micro electrode. The obtained 80
µm needle was further tapered down to 10 µm. Afterward, gold was deposited on either side of the glass probe and a Parylene insulating layer was formed on top of the gold layer. Finally, the recessed tip on the micro electrode was formed by HF etching (Fig. 8a). The needle type micro electrode showed a linear sensitivity of 147 pA/mg.L-1. The conventional DO micro electrode
was less sensitive with a slope of only 10 pA/mg.L-1. The needle type micro electrode was used
to study the DO-profile in a bacterial biofilm. The DO in the bulk solution was 8.5 mg.L-1,
which decreased to 5.9 mg.L-1 at the surface of the biofilm. At 700 µm inside the biofilm, no
