공학박사학위논문

Development of Electrical Conductivity Measurement Technology for Key Plant

Physiological Information using Microneedle Sensor

마이크로바늘 센서 이용 식물체 주요 생체정보(전기전도도)

측정 기술 개발

2018 년 02 월

서울대학교 융합과학기술대학원

나노융합학과

전 은 용

마이크로바늘 센서 이용 식물체 주요 생체정보

(전기전도도) 측정 기술 개발

Development of Electrical Conductivity Measurement Technology for Key Plant Physiological Information using

Microneedle Sensor

지도교수 이 정 훈

이 논문을 공학박사 학위논문으로 제출함

2018 년 1 월

서울대학교 융합과학기술대학원

나노융합학과

전 은 용

전은용의 공학박사 학위논문을 인준함

2018 년 1 월

위 원 장 : 고 상 근 (인)

부위원장 : 이 정 훈 (인)

위 원 : 김 성 재 (인)

위 원 : 안 지 훈 (인)

위 원 : 조 일 환 (인)

i

Abstract

Development of Electrical Conductivity Measurement Technology for Key Plant Physiological Information using Microneedle Sensor

Eunyong Jeon

Nano Science and Technology Graduate School of Convergence Science Technology

Seoul National University

In plant cultivation, electrical conductivity measurement systems have been used

to manage salinity of nutrient solutions and soil (growing medium). Recent research

has shown that the quality and quantity of horticultural crops, e.g., cucumber and

tomato, can be optimized by controlling the salinity of nutrient solutions. However,

the continuous interplay between cultivars, electrical conductivity of nutrient

solution, greenhouse environment and cultivation practices are quite complex and

less understood. Understanding the detailed response of a plant to a nutrient solution

is not possible until the fruit is fully grown or by sacrificing the stem. In order to

overcome this problem, a plant internal information (electrical conductivity)

measuring device through real-time monitoring is needed.

In this study, I propose a microneedle probe system to measure plant internal

information. The xylem and phloem are organs that transport nutrients and water in

plants. The microneedle sensor measures the electrical conductivity of sap flowing

through xylem and phloem respectively. It is intended to measure the concentration

of water and minerals that plants absorb from their roots and the ionic changes due

to internal action. If plant internal physiological information and existing external

measurement information are integrated, plant growth environment optimization can

be achieved through efficient salinity management.

The fabrication process of the microneedle sensor includes silicon-based

ii

techniques such as microscale deposition, photolithography and deep etching

processes. Further, microscale fabrication enables all functional elements to be

within area budget and be very accurate with minimal plant invasion. A single unit

of xylem and phloem is in microscale. Therefore, the internal measurement space is

limited and measurement error may occur depending on the fringe effect.

Interdigitated electrodes have short current path between the electrodes and are

suitable for small size sensing platforms due to efficient arrangement of electrode

areas. The distance and width between the electrodes were designed by calculating

the cell constant and evaluated using an impedance measurement device. In addition,

microneedle sensor has electrode array on the needle tip. It has been designed to

measure a specific xylem or phloem inside a plant stem. Experiments were

conducted to confirm the necessity of the array sensor in the plant stem and it was

proved by the results. In order to access individual xylem and phloem, tomato was

used.

There are several important factors to be considered when developing a system

that can be inserted into plants. First, the electrode should be separated from the inner

tissue of the plant to avoid interference from measurement of the electrical

conductivity of the sap in the plant stem. The electric conductivity measurement is

undervalued when metal electrodes comes into contact with the internal structure of

the plant due to the fringe effect with the electric double layer formed on the

electrode surface. In this study, I proposed an insulation structure to protect the

electrodes, and designed and tested it through simulation.

Second, it is required to enhance the sensor performance by maximizing the

electrode surface area. The active electrode area is related to the optimization of the

iii

measuring range and the reduction of the polarization phenomenon. In this study, I

propose a sensor optimization by increasing the measurement range and reducing the

polarization phenomenon by forming a gold nanorod (GNR) on the electrode surface.

The developed microneedles were integrated with a measurement system based

on an impedance analysis chip. The converted digital signal from the measurement

module communicated with computer using Bluetooth technology via the

microcontroller. The developed device was inserted into a cucumber grown in a

greenhouse to confirm the possibility and analyze the measured signal. This device

is designed to be applied to horticultural crops (paprika, tomato, etc.). However, this

study focuses on analyzing basic data using cucumber. Micro needle sensor

technology measures internal plant physiological information that has not been

measured so far. Immediate monitoring of plant response data can create an effective

growth environment and increase yield and improve the quality of corp.

Keywords: Electrical conductivity, microneedle, real-time monitoring, impedance,

minimum invasive, plant cultivation

Student number: 2009-23796

iv

Contents

Abstract ··················································································· ⅰ

Contents ··················································································· ⅳ

List of Figures ············································································ ⅷ

List of Tables ·········································································· ⅹⅳ

Chapter 1. Introduction ································································· 1

1.1 Plant cultivation in greenhouse ··················································· 1

1.2 Real-time monitoring measurement system in plant stem ····················· 3

1.3 Electrical conductivity in plant cultivation ······································ 4

1.4 Microneedle electrical conductivity monitoring sensor ························ 5

1.5 Electrical measurement in microscale needle probe ··························· 6

1.6 References ··········································································· 7

Chapter 2. Electrical conductivity ··················································· 10

2.1 Electrical conductivity measurement theory ·································· 10

2.1.1 Electrical conductivity ······················································· 10

2.1.2 2-pole electrode system ······················································· 11

2.1.3 4-pole electrode system ······················································ 13

2.2 Electrical conductivity measurement system for microneedle probe ······ 15

2.2.1 Electrical conductivity system for plant application ······················ 15

v

2.2.2 Impedance circuit design ······················································ 16

2.3 Cell constant ······································································ 20

2.3.1 Cell constant modeling and calculation ···································· 20

2.3.2 COMSOL simulation analysis ················································ 24

2.4 Conclusion ········································································ 28

2.5 References ········································································· 29

Chapter 3. Microneedle probe fabrication and method ························· 30

3.1 Microneedle probe ······························································· 30

3.2 Fabrication process and Sensor inspection ···································· 32

3.2.1 Pre-test for needle fabrication ··············································· 32

3.2.2 Fabrication process for microneedle sensor ······························· 36

3.2.3 Sample inspection ···························································· 39

3.3 Microneedle sensor frequency characteristic evaluation ···················· 41

3.3.1 Frequency sweeping results ················································· 41

3.3.2 Frequency sweeping results (geometry variation) ························ 45

3.4 SU-8 support structure ··························································· 47

3.4.1 The interference between plant tissue and electrodes ···················· 47

3.4.2 Sample fabrication and materials ··········································· 49

3.4.3 Simulation and experiment results ·········································· 51

3.5 Conclusion ·········································································· 54

3.6 References ········································································· 55

Chapter 4. Measurement system ···················································· 57

4.1 Electrical conductivity measurement system overview ······················ 57

vi

4.2 Device operation and calibration ··············································· 60

4.3 System evaluation ································································ 63

4.3.1 Signal to Noise Ratio (SNR) of device ····································· 63

4.3.2 Impedance characteristics (AD5933 integrated sensor) ·················· 65

4.3.3 Real-time impedance monitoring ··········································· 68

4.3.4 Standard solution calibration ················································ 70

4.3.5 Temperature effect evaluation ··············································· 73

4.3.6 Array sensor evaluation ······················································ 75

4.4 Conclusion ·········································································· 80

4.5 References ········································································· 81

Chapter 5. Real-time monitoring measurement for electrical conductivity inside

of cucumber stem ······································································· 82

5.1 Cucumber cultivation for experiment ··········································· 82

5.2 Relative temperature in the plant stem ·········································· 84

5.2.1 Relative temperature measurement methodology ·························· 84

5.2.2 Relative temperature in cucumber stem ····································· 85

5.2.3 Correlation between external temperature and relative temperature ····· 86

5.3 Plants defense mechanism ······················································ 88

5.4 Cucumber electrical conductivity monitoring ·································· 91

5.4.1 Electrical conductivity of plant sap ········································· 91

5.4.2 Real-time change of electrical conductivity in plant stem ················ 91

5.4.3 Cucumber monitoring data ···················································· 94

5.5 Conclusion ·········································································· 99

vii

5.6 References ········································································· 100

Chapter 6. Optimization and enhancement ········································ 102

6.1 Gold Nano Rod (GNR) array on electrode ···································· 102

6.1.1 Enhanced sensor performance by surface modification ·················· 102

6.2 GNR array fabrication and results ·············································· 105

6.2.1 GNR array fabrication on electrode ······································· 105

6.2.2 Fabrication results ··························································· 107

6.2.3 Test results ···································································· 110

6.3 Conclusion ······································································· 113

6.4 Reference ········································································· 114

Chapter 7. Conclusion ································································ 116

Appendix ················································································· 120

A. Fabrication method for nanostructures ········································ 120

A1. Anodized aluminum oxide ··················································· 120

A2. Gold nanorod ·································································· 122

B. LabVIEW script for anodization process ······································ 123

C. LabVIEW script for frequency sweep measurement ························· 125

D. Appendix reference ······························································ 127

Abstract in Korean ···································································· 128

viii

List of Figures

Chapter 1. Introduction

Figure 1-1. Brief summary of protected cultivation

Figure 1-2. The concept of microscale needle probe system

Chapter 2. Electrical conductivity

Figure 2-1. Electrical conductive cell & measurement method

Figure 2-2. 2-pole conductivity measurement system

Figure 2-3. 4-pole conductivity measurement system

Figure 2-4. Theoretical study; equivalent circuit system of 2-pole cell

Figure 2-5. Theoretical study; equivalent circuit system of 2-pole cell (simplified)

Figure 2-6. Concept of impedance variation according to frequency change

Figure 2-7. Three-dimensional geometry of the interdigitated electrode; schematics

of the electrode structure and its dimensions (H: height, W: width, D: inter-distance

between two electrodes

Figure 2-8. Parallel plate capacitor test model; metal radius: 10cm, separation

distance: 3 cm, air domain radius: 20 cm, glass quartz relative permittivity: 4.2

Figure 2-9. Computing the effect of fringing fields on capacitance; metal radius: 10

cm, separation distance: 3 cm, air domain radius: 15 cm (initial sphere radius), glass

quartz relative permittivity: 4.2, parametric study of air domain radius: 15-39 cm

Figure 2-10. Computing the effect of fringing fields on capacitance (result)

Figure 2-11. Computing the effect of fringing fields on capacitance and cell constant

evaluation for interdigitated electrode

Figure 2-12. The interdigitated electrode computation results

ix

Chapter 3. Microneedle probe fabrication and method

Figure 3-1. Cucumber cross-section with prototype of sensor; cucumber stem and

microneedle integrated with PCB

Figure 3-2. Simplified sensor structure; micro-fabricated needle structure with

interdigitated array and electrode pad

Figure 3-3. Fabrication process of silicon microneedle

Figure 3-4. Fabricated silicon microneedle; wafer scale (left), single needle (right)

Figure 3-5. Fabricated silicon microneedle inserted into plant stem

Figure 3-6. Fabrication process of the microneedle electrical conductivity sensor: (a)

first passivation layer deposition, (b) gold electrode patterning, (c) second

passivation layer deposition, (d) passivation layer dry etch, (e) PR mask coating and

patterning, (d) front DRIE etching

Figure 3-7. MEMS processed microneedle sensors (wafer scale)

Figure 3-8. Fabricated microneedle sensor inspection setup and equipment

Figure 3-9. Initial impedance variation of fabricated microneedle sensors

Figure 3-10. Initial impedance variation of microneedle sensor after wire-bonding

Figure 3-11. Impedance measurement setup; frequency sweep 1 kHz to 2 MHz via

LabVIEW program

Figure 3-12. Frequency sweep results; filled circle: 0.001 wt% NaCl, empty

rectangular: 0.01 wt% NaCl, star: 0.1 wt% NaCl, empty circle: 0.3 wt% NaCl

Figure 3-13. Frequency sweep result; frequency sweep range 2 Hz to 2 MHz,

electrode geometry (W5S10, W3S2)

Figure 3-14. COMSOL-multiphysics electrical potential (V) simulation study for

planar electrode

x

Figure 3-15. Patterned SU-8 dry film on microneedle sensor

Figure 3-16. SU-8 applied microneedle fabrication process

Figure 3-17. COMSOL multiphysics simulation result; electrical field scan from

electrode to y-axis

Figure 3-18. Real-time measurement to evaluate effect of SU-8 support structure

using hydrogel; normal (unprotect) electrode test result (graph above), SU-8 applied

electrode test result (graph below)

Figure 3-19. Conceptual diagram of SU-8 supported electrode and unprotected

electrode; unprotect electrode (left), protected electrode (right)

Chapter 4. Measurement system

Figure 4-1. AD5933 12-bit impedance converter; functional block diagram and chip

configuration

Figure 4-2. Microneedle probe electrical conductivity measurement system

integrated with AD5933; (a) microneedle chip integration with PCB using wire

bonding (b) microneedle impedance analyzer package

Figure 4-3. Schematic of microneedle measurement device and details of relay

system for AD5933 isolation

Figure 4-4. System operation schematic with AD5933 evaluation board and

microcontroller (Arduino)

Figure 4-5. Magnitude signal variation via calibration resistance change in AD5933

system

Figure 4-6. System module calibration sequence: 1) pick target resistor, 2) measure

gain and magnitude with various known resistors, 3) calculate the noise of

xi

impedance, 4) define target range

Figure 4-7. Measurement system noise evaluation setup

Figure 4-8. Measurement system noise evaluation with high precision resistor

(without ground isolation system)

Figure 4-9. Measurement system noise evaluation with high precision resistor (with

ground isolation system)

Figure 4-10. Frequency sweep test result; impedance measurement module

integrated with AD5933 impedance analyzer chip

Figure 4-11. Real-time monitoring result of concentration change due to additional

supply of concentrated solution

Figure 4-12. Microneedle electrical conductivity sensor calibration using various

concentration of standard solution (0.084 ~ 12.880 mS/cm, linear fitting)

Figure 4-13. Three sets of calibration curves; Three different microneedle sensor

evaluated by standard solution

Figure 4-14. Test setup for temperature effect on microneedle sensor. A Teflon tube

was used to create a plant vesicular bundle like environment

Figure 4-15. Test results for temperature effect on sensor

Figure 4-16. Illustration of microneedle array sensor schematics and sensor package

Figure 4-17. The experiment setup and details of microneedle: (a) experiment setup

at lab scale approach, (b) experiment setup and test period for greenhouse, (c) the

microneedle sensor parametric details for needle insertion

Figure 4-18. Microneedle array sensor test result (at night 20:54 ~ 21:38)

Figure 4-19. Microneedle array sensor test result (at day 13:58 ~ 15:07)

Figure 4-20. Real-time monitoring sensor system with microneedle sensor

xii

Chapter 5. Real-time monitoring measurement for electrical conductivity inside

of cucumber stem

Figure 5-1. Greenhouse cucumber cultivation and real-time monitoring experiment

Figure 5-2. Temperature change in and outside of cucumber stem: relative

temperature (straight line), ambient temperature monitored by temperature sensor

integrated with AD5933 (dotted line)

Figure 5-3. Correlation between internal relative temperature and external relative

temperature. The linear correlation was observed

Figure 5-4. Schematic of the plant defense mechanism for wound

Figure 5-5. Schematic of the plant defense mechanism effect to needle insertion

Figure 5-6. Schematic of the slime coated needle (left) and interdigitated electrode

(right)

Figure 5-7. Schematic of the needle installation with sealing technique

Figure 5-8. Cucumber plants grown in a greenhouse environment for experiment.

Cucumbers used in the experiment are indicated by black arrows

Figure 5-9. The real-time monitoring electrical conductivity measurement result;

immediate observation of changes in electrical conductivity value reflecting external

environmental changes

Figure 5-10. Microneedle sensor inserted in cucumber stem

Figure 5-11. The real-time monitoring electrical conductivity measurement result;

sensor 1 (mS/cm) at ambient temperature (degree Celcius)

Figure 5-12. The real-time monitoring electrical conductivity measurement result;

sensor 2 (mS/cm) at ambient temperature (degree Celcius)

Figure 5-13. The real-time monitoring electrical conductivity measurement result;

xiii

sensor 3 (mS/cm) at ambient temperature (degree Celcius)

Figure 5-14. The real-time monitoring electrical conductivity measurement result;

sensor 4 (mS/cm) at ambient temperature (degree Celcius)

Chapter 6. Optimization and enhancement

Figure 6-1. Increase electrode surface area modifying GNR array on electrode; 109

single GNR fabricated on 1 µm x 1 µm area. Single GNR diameter is 70 nm

Figure 6-2. The schematic of frequency region according to electrode configuration

Figure 6-3. The schematic of frequency region changes according to increase

electrode surface area (Effect of EDLC modification)

Figure 6-4. Apparatus for oxidation process: (a) wafer scale processing zig made of

PEEK material with strong chemical resistance (b) internal cross-section of

equipment (c) elastic electrode to reduce contact resistance and protect sample

surface (d) ring electrode

Figure 6-5. Fabricated surface modified GNR sensor; diced GNR sample (left), 12.3

mm x 30k SEM image (upper right), 12.3 mm x 200k SEM image (lower right)

Figure 6-6. Interdigitated electrode integrated with GNR array; 12.3 x 35.0k SEM

image for GNR (upper), GNR on the interdigitated electrode (lower left), fabricated

sensor (lower right); arrow points out the imaged GNR on the chip

Figure 6-7. Frequency sweep result for planer interdigitated electrode. The unit of

the standard solution is µS/cm

Figure 6-8. Frequency sweep result for GNR modified interdigitated electrode. The

unit of the standard solution is µS/cm

Figure 6-8. Frequency sweep result for GNR modified electrode vs. Planar electrode.

xiv

The unit of the standard solution is µS/cm

Appendix

Figure A-1. Process setup for making aluminum oxide. The PEEK zig has chemical

resistance and is designed to be suitable for processes using strong acids. A voltage

is applied between the electrode inserted inside the zig and the electrode leading to

the electrolyte, and oxidation phenomenon occurs.

Figure A-2. (a) process flow for AAO and gold nanorod array (GNR). (b) AAO filled

by gold reduction reaction. (c) completed GNR after AAO template removal process

Figure A-3. Front panel for current measurement system (anodizing ver.)

Figure A-4. Block diagram for anodizing or electroplating system

Figure A-5. Front panel for impedance measurement system (frequency sweep ver.)

Figure A-6. Block diagram for impedance measurement system with sequential

incensement of frequency

List of Tables

Table 2-1. Electrical conductivity measurement range variation according to cell

constant value

Table 4-1. Frequency sweeping results with an AD5933 and measurement circuit

Table 4-2. Calibration results of standard solution with an AD5933 measurement

circuit

1

Chapter 1. Introduction

In this thesis, I present design and fabrication of real-time electrical conductivity

monitoring system: microscale needle probe and impedance measurement system

with relay. The sensor platform was inserted into the plant stem to optimize the

electrode and system for measurement. In addition, the laboratory-proven sensors

were applied to greenhouse cultivated cucumber and the electrical conductivity were

monitored for few days. Results indicate peculiar periodic pattern of ionic

concentration variation with respect to time and other factors. The plant's defenses

against external injuries have been studied and verified experimentally.

In addition, I suggested the electrode integrated nanoscale gold rod array that can

improve the frequency properties of impedance measurement systems to achieve

high reliability and measurement range.

1.1 Plant cultivation in greenhouse

The greenhouse cultivation is a high tech production facility developed to cope

with increasing demand for fruit and vegetables. It is also called protected cultivation.

Depending on the situation and conditions, various transparent materials such as

vinyl or glass are used to create the optimal cultivation environment. The growth

factors: humidity, temperature, mineral nutrient, light intensity, atmospheric

composition, and irrigation can be controlled. As a result of the controllable

cultivation environment, it is possible to extend the cultivation period without being

affected by the climate or the environment of the specific region. Also, the quality of

the crops can be improved and the yield can be increased by optimizing the growth

2

conditions [1-4].

In order to optimize and maintain the growth environment, a technology that can

monitor the ambient condition of the greenhouse is needed. Therefore, the

greenhouses are filled with equipment such as solar radiation, temperature, electrical

conductivity, carbon oxide, moisture sensor, to name a few. These facilities are

already commercialized in the field at the moment. However, there still exist many

limitations which need to be solved. The physiology of plants is very complex and

not well known. Establishment of growth conditions using environmental control

method requires long time investment and can only be analyzed at sacrifice of flesh

or plant stem. A reasonable solution to the problem is to measure and analyze in-

plant information in real time in response to changes in the external environment.

Figure 1-1. Brief summary of protected cultivation

3

1.2 Real-time monitoring measurement system in plant stem

Research using real-time monitoring methods to measure plant physiological

information has been active since the mid-19th century. The main object to be

measured was the fluid flow rate [5] and resistance [6] of the vesicular bundle inside

the plant stem consisting of xylem and phloem. These two component transport the

moisture and nutrients of the plant. Such functions make the vesicular bundles the

most suitable part of plant internal information measurements because it is directly

related to transpiration and photosynthesis which are very important for plant

physiology.

Methods for measuring fluid flow include heat pulse velocity method [7], heat

field deformation [8] and thermal dissipation probes [9]. All of these techniques

involve heating electrodes and predict the speed by measuring thermal conduction

according to the degree of sap flow in the plant stem. The flow rate of the plant sap

is the phenomenon caused by the evaporation of the plant, so the amount of water

evaporated is known. Therefore, it can be compared with the amount of nutrient

supplied and the absorption efficiency relative to the supply can be analyzed. The

resistance of plant internal fluid was used as an indicator of plant condition [10].

Electrical resistance is an important indicator of the state of the plant interior. The

principle of measurement is to measure the internal electrical conductivity by

inserting two probes into a plant and applying an electric pulse. Typically, a

shigometer is one of the representative devices for measuring plant health by

measuring electrical resistance. I use a method to measure resistance increase due to

internal oxide increase when a plant is infected or decayed. However, the fluid flow

and electrical resistance measuring devices developed so far had to incorporate

4

invasive probes into the plant and additionally required large and heavy measuring

modules. It can be applied to woody plants such as trees, but it is not applicable to

plants with thin stems and fragile plants such as horticultural crops.

1.3 Electrical conductivity in plant cultivation

It is very important to manage the salinity of the nutrient solution using the electric

conductivity in the greenhouse cultivation facility. The following are important salts

for growing plants; Na+, Mg2+, Cl-, SO42- and HCO3

-. Salinity is expressed in terms

of electrical conductivity (mS/cm) depending on the degree of current flow in the

solution, which varies with the concentration of ions. The excess or deficiency of the

salinity will negatively affect the growth of the plant. For example, Salt stress in

cucumber cultivation has a negative effect on the overall growth; the higher the value,

the greater is the effect [11-12]. In the case of tomatoes, increasing the salinity of the

nutrient solution increases the ion concentration of the soil more than the inside of

the plant, and the osmotic potential drastically decreases. There is a lack of water

transport, which has a relatively positive effect on the quality of the fruit, but there

is a considerable loss in terms of yield [13-14].

Currently, in the horticulture cultivation environment, salinity management is

indirectly supported by the supply system. The electrical conductivity of the nutrient

solution is compared with the value of the solution discharged from the block

(soilless, cocopeat). Salinity values can increase over time as ions accumulate in the

block. In order to establish the correct nutrient supply and plant growth conditions,

information on plant ion concentration is needed. The internal physiological

information reflects the state of the plant and can be used to effectively manage the

horticulture cultivation in real time.

5

1.4 Microneedle electrical conductivity monitoring sensor

To measure the real-time data of such thin and weak plants, I propose a

microneedle sensor having a small form factor, with minimal invasion. The needle

structure can effectively reach the vascular bundle while minimally invading the

plant stem. However, considering that the size of the vascular tissue is several

hundreds of micro-scale, the needles must also implement microscale and electrodes

must be formed on the needles to measure electrical conductivity. It is well known

field of drug delivery and neuroscience that microneedle has scaling effect and

functionality [15]. Microelectromechanical systems technology was introduced to

fabricate micro needle sensors. Semiconductor processes were optimized for making

micro-scale structures. Using a photolithographic process, an electrode with a size

of several tens of micrometers can be fabricated. The fabricated sensor is connected

to the printed circuit board through packaging.

Figure 1-2. The concept of microscale needle probe system.

6

1.5 Electrical measurement in microscale needle probe

It has structural and functional advantages by using Micro scale needle structure.

However, in terms of the electrical conductivity measurement system, the

measurement range is limited due to the reduction of the electrode area. Therefore,

it is necessary to maximize the surface area by any method. Therefore, the most

commonly used method is to use platinized electrodes [16]. However, in this case,

there is a limit to the electrode material and it is difficult to apply to the MEMS

process. In this study, nanostructures were formed on the electrode surface in array

form to maximize the surface area and to solve the polarization phenomenon. AAO

is a porous oxide film with nano-sized holes. The feature of this material is the

versatility to form thicknesses from hundreds of nano to microscale. It is also used

to fabricate two-dimensional structures such as nanorods because nanoscale holes

are created vertically [17]. Some researchers have used AAO for semiconductor

processing [18]. I fabricated a nanorod array by applying nanofabrication technology

to micro needle sensor electrodes. A special oxide film manufacturing system was

fabricated and applied to the MEMS process, confirming the formation of nanorods

on the electrodes. Planar and nanorods modified electrodes were next tested and

nanorods modified electrodes were observed to have better performance.

7

1.6 References

[1] Katerji, N., Van Hoorn, J. W., Hamdy, A., & Mastrorilli, M. (2003). Salinity effect

on crop development and yield, analysis of salt tolerance according to several

classification methods. Agricultural water management, 62(1), 37-66.

[2] Romero-Aranda, R., Soria, T., & Cuartero, J. (2001). Tomato plant-water uptake

and plant-water relationships under saline growth conditions. Plant Science, 160(2),

265-272.

[3] Grattan, S. R., & Grieve, C. M. (1998). Salinity–mineral nutrient relations in

horticultural crops. Scientia horticulturae, 78(1), 127-157.

[4] Lechno, S., Zamski, E., & Tel-Or, E. (1997). Salt stress-induced responses in

cucumber plants. Journal of Plant Physiology, 150(1-2), 206-211.

[5] Marshall, D. C. (1958). Measurement of sap flow in conifers by heat

transport. Plant physiology, 33(6), 385.

[6] Tattar, T. A., & Blanchard, R. O. (1976). Electrophysiological research in plant

pathology. Annual Review of Phytopathology, 14(1), 309-325.

[7] Swanson, R. H., & Whitfield, D. W. A. (1981). A numerical analysis of heat pulse

velocity theory and practice. Journal of experimental botany, 32(1), 221-239.

[8] Nadezhdina, N., Vandegehuchte, M. W., & Steppe, K. (2012). Sap flux density

measurements based on the heat field deformation method. Trees, 26(5), 1439-1448.

[9] Lu, P., Urban, L., & Zhao, P. (2004). Granier's thermal dissipation probe (TDP)

method for measuring sap flow in trees: theory and practice. Acta botanica sinica

englich edition, 46(6), 631-646.

8

[10] Shigo, A. L., & Shigo, A. (1974). Detection of discoloration and decay in living

trees and utility poles.

[11] Duan, J. J., Guo, S. R., Kang, Y. Y., Li, J., & Liu, X. E. (2008). Effects of salt

stress on cucumber seedlings root growth and polyamine metabolism. Ying yong

sheng tai xue bao= The journal of applied ecology, 19(1), 57-64.

[12] Khan, M. M., Al-Mas' oudi, R. S., Al-Said, F., & Khan, I. (2013). Salinity effects

on growth, electrolyte leakage, chlorophyll content and lipid peroxidation in

cucumber (Cucumis sativus L.). International Proceedings of Chemical, Biological

& Environmental Engineering, 55(6), 28-32.

[13] Mitchell, J. P., Shennan, C., & Grattan, S. R. (1991). Developmental changes in

tomato fruit composition in response to water deficit and salinity. Physiologia

Plantarum, 83(1), 177-185.

[14] Mitchell, J. P., Shennan, C., Grattan, S. R., & May, D. M. (1991). Tomato fruit

yields and quality under water deficit and salinity. Journal of the American Society

for Horticultural Science, 116(2), 215-221.

[15] Prausnitz, M. R. (2004). Microneedles for transdermal drug delivery. Advanced

drug delivery reviews, 56(5), 581-587.

[16] Feltham, A. M., & Spiro, M. (1971). Platinized platinum electrodes. Chemical

Reviews, 71(2), 177-193.

[17] Evans, P., Hendren, W. R., Atkinson, R., Wurtz, G. A., Dickson, W., Zayats, A.

V., & Pollard, R. J. (2006). Growth and properties of gold and nickel nanorods in

thin film alumina. Nanotechnology, 17(23), 5746.

9

[18] Kim, Y., Jung, B., Lee, H., Kim, H., Lee, K., & Park, H. (2009). Capacitive

humidity sensor design based on anodic aluminum oxide. Sensors and Actuators B:

Chemical, 141(2), 441-446.

10

Chapter 2. Electrical conductivity

2.1 Electrical conductivity measurement theory

2.1.1 Electrical conductivity

Electrical conductivity is a numerical representation of the degree to which current

flows through a material. In case of metal, current is conducted via the movement of

free electrons, but in electrolytes, current is generated by the movement of positive

or negative ions. The electrical conductivity of a solution can be measured by voltage

difference generated by applying an alternating current between two electrodes. In

this process, the cations move to the negative electrode, the anions move to the

positive electrode, and the solution acts like a conductor.

Figure 2-1. Electrical conductive cell & measurement method

11

Eq. (2-1) shows the relationship between the electrical conductivity (σ ), solution

resistance ( R ), and cell constant (κ ). The cell constant can be expressed by the

relationship between the working area of the electrode ( a ) and the inter-distance

between the electrodes ( d ), as shown in Eq. (2-2). The electrical conductivity can

be inversely calculated by obtaining the resistance of the plant sap, for an electrode

with a specific geometry.

=Rκσ (2-1)

= da

κ (2-2)

Several electrical conductivity measurement methods have been developed

depending on the measurement cell design. The 2-pole and 4-pole measurement

systems are most commonly used. There are advantages and disadvantages to each

method, depending on the environment you use.

2.1.2 2-pole electrode system

A system using two electrodes measures the potential difference between two

electrodes caused by the applied alternating current in Figure 2-2. The target

measurement element is the resistance of the electrolyte. However, the resistance

( R electrode ) generated by the polarization of the electrode surface can be measured

12

along with the target resistance ( R solution ), and an error may occur. Therefore, a

calibration process using a standard solution is required. An important factor in this

system is the cell constant. The cell constant is the ratio of electrode distance to active

electrode area. The higher the concentration of the sample, the larger the cell constant

is required. The cell constant selection criteria is shown in Table (2-1).

The shape of the 2-pole electrode does not limit the generated current path. During

the measurement, current flows in all directions and this current distribution is

influenced by the electrical properties of the solution (electrical permittivity, ε) and

the chamber wall or floor being measured [1]. Therefore, more accurate value can

be obtained by always measuring at the same position. Despite these disadvantages,

the 2-pole electrode configurations continue to be used because of their relatively

simple composition and sensitivity in low-concentration ranges.

Table 2-1. Electrical conductivity measurement range variation according to cell constant value [2].

13

Figure 2-2. 2-pole conductivity measurement system

2.1.3 4-pole electrode system

The 2-pole electrode cell has a simple structure and can be used in a various

environment, from very low concentration up to about 100 mS/cm [2]. However, in

a solution having a high ion concentration, a current reduction due to mutual

repulsion of ions occurs, called polarization phenomenon. This configuration is

therefore difficult to use in certain applications where very high ion concentrations

are to be measured. The 4-pole electrode cell contains two electrodes for the current

path and two electrodes for measuring the potential shown in Figure 2.3. Polarization

does not occur at the electrode that only measures the voltage. Therefore, accurate

measurement is possible even at high ion concentration. Yet, the design is

14

complicated compared to the 2-pole electrode.

Figure 2-3. 4-pole conductivity measurement system

15

2.2 Electrical conductivity measurement system for microneedle probe

2.2.1 Electrical conductivity system for plant application

The microneedle probe sensor has an array sensor platform. Considering the

thickness of the needle and the electrode width and spacing, the 4-pole cell system

is difficult to apply. Therefore, a 2-pole electrode method was used. Several

considerations have been taken into account for the intended use of the measurement.

The first consideration in designing an electrode for use in plants is setting the

measurement range. As mentioned in the introduction part, nutrient solution with

electrical conductivity value higher than 10 mS/cm is not used for horticultural crop

cultivation such as cucumber or tomato. Therefore, the measurement range is set to

0 ~ 20 mS/cm considering the safety factor. The second consideration is electrode

type determination for the measurement environment. Scale effects such as minimal

invasion and noise reduction can be expected by applying a microscale needle probe.

However, efficient electrode design must be applied as available electrode area is

limited. The third is the stability of the sensor. The microneedle sensor measures

electrical conductivity inside plant stems, especially in vesicular bundle. The current

path must be localized and short to avoid possible interference and noise in the

measuring area.

Considering the above, the interdigitated electrode is an appropriate solution. The

interdigitated electrode configurations are universally used in impedance

spectroscopy because of their high sensitivity and general utility [1]. Due to the

efficient arrangement of the electrodes, the gap between the electrodes can be

reduced and the electrode area can be widened. Compared to the conventional 2-pole

16

electrodes, the system can achieve relatively low cell constants. Therefore, the range

of measurement applicable to plants can be attained. In addition, the current path is

very short and wide. The accuracy can be increased by reducing the noise due to the

structural environment when measuring inside the plant.

2.2.2 Impedance circuit design

In this research, interdigitated 2-pole system was used for microneedle probe.

These electrodes are made up using equal geometric surface area. The parallel

electrodes are facing each other and separated by a gap. The equivalent circuit in

such a structure can be expressed as Figure 2-4. In Figure 2-4, each component is

indicated by solution resistance ( SOLR ), solution dielectric capacitance ( EC ), and

the electrical double layer capacitance ( EDLC ) at the solution-electrode interface [3].

Since the EDLC generated at the electrolyte/electrode interface of both electrodes

are in parallel connection, the capacitances can be simplified as an equivalent

capacitance shown in Figure 2-5. However, this assumption may not be true for very

high resistance solutions (low conductivity solutions) or alternating current systems

using very high frequencies [3].

17

Figure 2-4. Theoretical study; equivalent circuit system of 2-pole cell

Figure 2-5. Theoretical study; equivalent circuit system of 2-pole cell (simplified)

The overall impedance (Z) of this circuit in response to sinusoidal voltage input

can be represented by Eq. (2-3). The magnitude of Z, ( )Z ω can be calculated

using Eq. (2-3). At low and high frequencies, ( )Z ω can be approximated using

Eq. (2-7) and (2-8), respectively.

18

2

1( ) ( )( ) ( )

SOL EDL

SOL E EDL EDL E

R C jVZI R C C j C C j

ωω ωω ω

+= =

+ + (2-3)

2( ) ( ( ))Z Zω ω= (2-4)

2 2 2

2 2 2 4 2

1( )( )

SOL EDL

SOL E EDL EDL E

R CZR C C C C

ωωω ω

+=

+ + (2-5)

2

,

ff

ff

ω π

ωω

ω

∴ =

↑⇒ ↑∝

↓⇒ ↓ (2-6)

2

1,

( )

1( ) , lim ( ) 0

SOL EDL

SOL E EDL

highE

R CZR C C

Z ZC ω

ωωωω

ω ωω →∞

>>

=

∴ = =

(2-7)

19

2

00

1,1 1( )

( )( )1( )

( )1 1( ) limlim ( )

EDL EEDL E

lowEDL E

EDL E

ZC CC C

ZC C

ZC C ωω

ω

ωωω

ωω

ωω ω→→

<<

= =++

∴ =+

= = ∗ = ∞+

(2-8)

It should be noted that EDLC is usually much larger than EC owing to the

nanoscale-thick electrical double layer (EDL) formed across the contact area

between the gold electrodes and the solution. These approximations indicate that the

impedance is dominated by the EDL at low frequencies, and by the solution dielectric

property at high frequencies. None of these extremes correlates with the solution

conductivity that I wish to measure. Throughout the course of frequency sweeping,

the impedance varies from EDL-dominated region to dielectric-dominated region.

The transient region between the extremes offers the window for the impedance

dependence on the solution resistance representing the EC value.

( )Z ω is shown in Figure 2-6. There are three regions according to frequency:

the first is the EDL capacitance dominant region ( EDLC ), the second is the solution

resistance dominant region ( SOLR ), and the last is the solution capacitance dominant

region ( EC ). Each region is divided based on the frequency (cut-off frequency) (fc)

described in Figure 2-6.

20

Figure 2-6. Concept of impedance variation according to frequency change

2.3 Cell constant

2.3.1 Cell constant modeling and calculation

In the measurement of electrolyte impedance using interdigitated electrodes, the

electrochemical cell constant ( cellκ ) is an important factor for determining the

measurement specification that depends on electrode geometry [4-7]. The cell

constant of two parallel electrodes can be determined using Eq. (2-9), where D and

A are the inter-electrode distance and the area of the electrode, respectively.

21

=cellDA

κ (2-9)

r o

cell

C ε ε=κ

(2-10)

Figure 2-7 shows the geometric parameters of the interdigitated electrodes, where

D is the inter-electrode distance, L is the finger length, and W and H are the electrode

width and thickness, respectively. The cell constant can be defined using the

capacitance of the measuring electrode (C), relative permittivity ( rε ), and

permittivity of vacuum ( οε ), as shown in Eq. (2-10). The COMSOL (ver. 5.2)

simulation tool was used to predict the capacitance of the measuring electrode. In

this study, I considered an interdigitated electrode structure with a width of 5 µm, a

finger length of 138 µm, electrode spacing of 10 µm, and total number of fingers of

14. The electrode structure was placed on the silicon microneedle protected by

silicon dioxide. Therefore, the thickness and material properties of the microneedle

and the protective layer were considered to simulate capacitance. The capacitance of

the electrode was simulated using COMSOL (ver. 5.2) and a cell constant value of

10.28 /cm, which was calculated from Eq. (2-10), was used. The values obtained

were compared to those reported by Olthuis et al. [5]. The fringing effect of the

electric field also needs to be taken into account when the spacing between the planar

electrodes is large for accurate consideration of the effective area. The overall

capacitance, the cell constant ( cellκ ) of planar interdigitated electrodes with the

22

given parameters, can be expressed as in Eq. (2-11).

2

2 ( )=( 1) ( 1 )

cellK k

N L K kκ ⋅

− − (2-11)

cos( )2

WkD W

π= ⋅

+ (2-12)

( )K k in Eq. (2-11) is the complete elliptic integral of the first kind to calculate the

effect of the fringing field, and N is the number of fingers. Modulus ( k ) can be

defined by Eq. (2-12) when the number of fingers is greater than two (N > 2). The

calculated cell constant value of the above-mentioned geometry using Eq. (2-11) is

12.967 /cm. The cell constants obtained using the COMSOL simulation tool and Eq.

(2-11) are slightly different. This is because in Eq. (2-11), the fringe effect between

the number N of fingers is considered, which results in a cell constant that is lower

than that obtained by considering the total electrode area.

23

Figure 2-7. Three-dimensional geometry of the interdigitated electrode; schematics of the electrode structure and its dimensions (H: height, W: width, D: inter-distance between two electrodes

24

2.3.2 COMSOL simulation analysis

The relationship between cell constant and capacitance is shown in Eq. (2-9 and

2-10). The capacitance of the electrode can be predicted and the cell constant can be

obtained inversely. The COMSOL multiphysics tool can simulate various physical,

chemical and mechanical phenomena. Interdigitated electrodes were reproduced in

three dimensional space and their capacitance was analyzed. In order to check

whether the model set up in this study is working, parallel plate electrodes that can

be easily analyzed were evaluated first. Figure 2-8 shows the parallel flat capacitor

used for modeling. The capacitance of the capacitor derived from the simulation

result were 43.429 pF. Compared to the calculated value of 38.941 pF with a simple

hand calculation, the simulation result shows higher value. Difference in numerical

results are relatively low because the fringe effect is not taken into account in the

hand calculations. Therefore, given the small difference, the simulation results are

reasonable.

Figure 2-8. Parallel plate capacitor test model; metal radius: 10cm, separation distance: 3 cm, air domain radius: 20 cm, glass quartz relative permittivity: 4.2

25

Figure 2-9. Computing the effect of fringing fields on capacitance; metal radius: 10 cm, separation distance: 3 cm, air domain radius: 15 cm (initial sphere radius), glass quartz relative permittivity: 4.2, parametric study of air domain radius: 15-39 cm

In order to improve the simulation accuracy, the boundary condition of the air

domain was set to zero charge and floating potential, and the average value of the

calculated values was used. Figures 2-9 and 2-10 show simulation model and study

results, respectively.

The capacitance values of the interdigitated electrodes were simulated based on

the previous modeling set up. The environment around the electrodes was set to

sodium chloride as in the actual experiment. Boundary condition was set to zero

charge, floating potential and average value was used.

Figure 2-11 shows detailed modeling information. The cell constants were

calculated using the value of the saturation point in the result of Fig. 2-12, and the

result was 10.28 / cm.

26

Figure 2-10. Computing the effect of fringing fields on capacitance (result)

Figure 2-11. Computing the effect of fringing fields on capacitance and cell constant evaluation for interdigitated electrode

27

Figure 2-12. The interdigitated electrode computation results

28

2.4 Conclusion

In this chapter, research was conducted to apply the electrical conductivity

measurement system to plant stem. The method of conductivity measurement and

the electrode type were determined taking into account the limited measurement

environment and the measurement window.

The two electrode measurement method was selected considering the electrode

installation area and the interdigitated electrode geometry was integrated to optimize

the area utilization and accuracy. Circuit analysis was carried out according to the

measurement method selection. You can understand the measurement system and

further estimate the actual measurement value. Two approaches were used to

calculate the cell constant. One is the capacitance method and the other is the

COMSOL simulation. The two methods were used to compare the values.

29

2.5 References

[1] Schiefelbein, S. L., Fried, N. A., Rhoads, K. G., & Sadoway, D. R. (1998). A

high-accuracy, calibration-free technique for measuring the electrical conductivity

of liquids. Review of scientific instruments, 69(9), 3308-3313.

[2] www.bat4ph.com/conductivity.html

[3] Electrolyte, D. (1999). Conductance Measurements Part 1: Theory. Current

Separations, 18(3), 92.

[4] Hong, J., Yoon, D. S., Kim, S. K., Kim, T. S., Kim, S., Pak, E. Y., & No, K. (2005).

AC frequency characteristics of coplanar impedance sensors as design

parameters. Lab on a Chip, 5(3), 270-279.

[5] Olthuis, W., Streekstra, W., & Bergveld, P. (1995). Theoretical and experimental

determination of cell constants of planar-interdigitated electrolyte conductivity

sensors. Sensors and Actuators B: Chemical, 24(1-3), 252-256.

[6] Ibrahim, M., Claudel, J., Kourtiche, D., & Nadi, M. (2013). Geometric

parameters optimization of planar interdigitated electrodes for bioimpedance

spectroscopy. Journal of Electrical Bioimpedance, 4(1), 13-22.

[7] Sheppard Jr, N. F., Tucker, R. C., & Wu, C. (1993). Electrical conductivity

measurements using microfabricated interdigitated electrodes. Analytical

Chemistry, 65(9), 1199-1202.

30

Chapter 3. Microneedle probe fabrication and method

3.1 Microneedle probe

Microneedle structures are predominantly utilized in the medical field because of

their advantages such as technical versatility and scaling effect [1–6]. Most

importantly, a microneedle structure enables measurement with minimum invasion.

In my plant-oriented application, the semiconductor process is used to fabricate

interdigitated electrodes on the microneedle structure, leading to precise impedance

measurement. The microneedle structure fabricated from a silicon wafer and the

cross-section of the cucumber stem with implanted microneedle sensor are shown in

Figure 3-1. Short protrusions are installed on either side of the sensing needle to

distribute the shear load due to the weight of the chip. The size of the complete device

is 11 mm × 5 mm, including the packaging base. The microneedle sensor is to be

inserted into the xylem of the plant stem to contact the fluid. The largest stem

diameters of the cucumber and tomato plants used in my experiments were 20 mm.

Previous studies have reported 5–20 mm stem diameters for tomato and cucumber

plants cultivated under a controlled growth environment [7-10]. Therefore, the

length of the needle was set to 5 mm to ensure penetration through the phloem and

access to the xylem. An array of sensing elements can be used to access the different

xylem locations. Figure 3-2 shows fabricated microneedle probe. The needle

thickness can be controlled by front and back deep reactive ion etching (DRIE)

process on the silicon wafer. The microneedle tip is designed to minimize the

penetration resistance during needle insertion. A sharp needle tip with 30° cone angle

has been shown to have low penetration resistance [11].

31

Figure 3-1. Cucumber cross-section with prototype of sensor; cucumber stem and microneedle integrated with PCB

32

Figure 3-2. Simplified sensor structure; micro-fabricated needle structure with interdigitated array and electrode pad

3.2 Fabrication process and Sensor inspection

3.2.1 Pre-test for needle fabrication

Prior to the preparation of the microneedle probe, preliminary experiments were

conducted to confirm the possibility of needle making and its suitability for plant

insertion. The length, width, and number of needles have been designed with as many

parameters as possible in consideration of future needle design changes. Deep

Reactive Ion Etching (DRIE) process technology was used to fabricate microscale

needles. DRIE process technology is used in special applications such as deep

punching with high anisotropic technology and fabricating structures with high

33

aspect ratio. This process requires a mask for selective etching. Usually, photoresist

(PR) is used as mask material for Reactive Ion Etching (RIE) and Inductively

Coupled Plasma (ICP) etching. However, in case of DRIE, the etching time is long

and the selectivity with PR is not enough to achieve full penetration of silicon wafer.

Aluminum was selected as the etching mask material. The selectivity ratio of

aluminum to the etching process is 5.44x10-3 nm / min [12]. The detailed process is

illustrated in Figure 3-3. The thickness of the needle was controlled by two etching

processes, front and back. The fabricated microneedle using this process is depicted

in Figure 3-4. The microneedle making process was established and the thickness

was controlled through the front and back DRIE process. However, according to the

results of the DRIE process, the uniformity in the wafer unit process drops. Overall

thickness variation occurred. This issue was overcome by one way etching process

from the bottom. The applied needle was applied directly to the plant. As shown in

Figure 3-5, insertion into the plant was easy. It was confirmed that it was inserted

without resistance when inserted. The number of needles has a great influence on

stability after insertion. Therefore, measurement needle and two auxiliary needle

structures were applied to improve the stability of micro needle sensor. In order to

prevent breakage in the trench of the device body and the needle, round processing

was performed.

34

Figure 3-3. Fabrication process of silicon microneedle

35

Figure 3-4. Fabricated silicon microneedle; wafer scale (left), single needle (right)

Figure 3-5. Fabricated silicon microneedle inserted into plant stem

36

3.2.2 Fabrication process for microneedle sensor

I used silicon etching and micro-patterning processes to fabricate the structure and

functional elements of the microneedle. Four-inch (100) double side polished P-type

boron doped silicon (1–10 Ω·cm) wafers were used for substrates. Silicon oxide film

(1 µm) was deposited with tetraethyl orthosilicate (TEOS) process (AMK, P-5000)

for the passivation layer. Electrodes were patterned with lift-off process after

patterning and the deposition of titanium (300 Å) and gold (2000 Å) with e-beam

evaporator (Maestech, ZZS550–2/D). Silicon oxide (1 µm) top passivation layer was

deposited with plasma enhanced chemical vapor deposition (PECVD) (Oxford

instruments, PlasmaPro System100), followed by patterning with photolithography

and the inductive coupled plasma (ICP) dry etching method (Oxford instruments,

PlasmaPro System100 Cobra).

Double step bulk micromachining was used for controlling the microneedle

thickness. Silicon wafer was etched from the top side by 200 µm with the DRIE

process (PLASMA-THERM, SLR-770-10R-B) using photoresist as a masking layer.

This process defined the shape and thickness of the microneedle. Aluminum (5000

Å) was deposited by sputtering (Sorona, SRN 120) for an etch stop layer. The silicon

substrate was then etched from the bottom side with the DRIE process to complete

the whole structure. The remaining aluminum layer and photoresist were removed

by wet etching and a dry etching process for release. The microneedle devices were

separated from the wafer by manually breaking the anchor parts.

37

Figure 3-6. Fabrication process of the microneedle electrical conductivity sensor: (a) first passivation layer deposition, (b) gold electrode patterning, (c) second passivation layer deposition, (d) passivation layer dry etch, (e) PR mask coating and patterning, (d) front DRIE etching

38

Figure 3-7. MEMS processed microneedle sensors (wafer scale)

39

3.2.3 Sample inspection

The initial impedance was measured to confirm the sample variation. For the

impedance measurement, a precision probe was connected to the E4980A impedance

analyzer shown in Figure 3-8. This study was carried out on 72 sensors manufactured

from two wafers in total, and one electrode pair was measured and compared for

each sensor.

Figure 3-8. Fabricated microneedle sensor inspection setup and equipment

The measured impedances were compared based on the total mean value. After

the process was completed, the post-packaging process were also compared because

it included the wire-bonding process. The overall sensor variation of the microneedle

sensor after the semiconductor process was confirmed to be 13% in Figure 3-9.

However, only 6% variation occurs when the sensor value corresponding to 80% of

40

the total impedance value was compared. On checking the sensor variation after wire

bonding, it was confirmed that the overall change was large. The sensor variation

was measured to be 14% and the variation of the sensor corresponding to 80% was

11% in Figure 3-10. It was confirmed that the initial impedance shift of the sensor

caused by wire bonding is large.

Figure 3-9. Initial impedance variation of fabricated microneedle sensors

41

Figure 3-10. Initial impedance variation of microneedle sensor after wire-bonding

3.3 Microneedle sensor frequency characteristic evaluation

3.3.1 Frequency sweeping results

As mentioned in Chapter 2, the frequency characteristic of the electrode is a way

of confirming the measurement window. It also serves as an important indicator for

determining the operating frequency. Therefore, the experiment was conducted to

analyze the frequency characteristics of the fabricated microneedle sensor. A sample

was prepared for frequency test measurement. All processes except DRIE were

applied. Needle feature were not implemented to reduce process costs and time

consumption. Diced samples were used to match electrode size. The sample had

42

same electrode shape as the micro needle sensor, so the results were acceptable. The

measurement setup for the experiment is depicted in detail in Figure 3-11. An LCR

meter (Agilent E4980A) was used to measure the impedance. The LCR meter was

connected to a computer using the GPIB interface, and the impedance value was

stored in text form using a program coded in LabVIEW.

The solution used in the experiment was sodium chloride. The purpose of this

experiment was to use an electrolyte solution that is directly related to salinity as

much as it measures the internal electrical conductivity of the plant. The solutions

0.001, 0.01, 0.1 and 0.3 wt% of NaCl analyte were prepared by diluting 2 wt% NaCl

stock solution. The prepared solution was sequentially injected into a

polydimethylsiloxane (PDMS) chamber fixed on the sensor electrode. In order to

reduce the measurement error, a predetermined volume was injected using a micro

pipette. The measured concentration increased gradually from low concentration,

and when replacing the solution, it was washed with ultrapure water.

Figure 3-12 graphs the impedance values for frequency changes from 1 kHz to 2

MHz. The higher the concentration, the lower the impedance is. The amount of

current flowing through the electrolyte increases while the impedance is low. For

each concentration, there is a section where the slope of the graph changes. The low

frequency band is the low cut off frequency and the high frequency band is the high

cut off frequency. The middle part of the two frequency ranges is the resistance

dominant area, and the impedance window according to the concentration difference

is the most substantial. The solutions of 0.001 wt% and 0.01 wt% show a low cut off

frequency range within 10 kHz. The low cut off frequency characteristics of 0.1 wt%

and 0.3 wt% of solution are shown above 100 kHz. In the case of high cut off

43

frequencies, the measurement limit is not measured beyond 2 MHz.

Figure 3-11. Impedance measurement setup; frequency sweep 1 kHz to 2 MHz via LabVIEW program

44

Figure 3-12. Frequency sweep results; filled circle: 0.001 wt% NaCl, empty rectangular: 0.01 wt% NaCl, star: 0.1 wt% NaCl, empty circle: 0.3 wt% NaCl

45

3.3.2 Frequency sweeping results (geometry variation)

The measurement system, using an LCR meter (Agilent E4980A), was operated

from 0–2 MHz, at 0.5 V, with geometric parameters of S=10, W=5, L=138, N=14

µm and S=2, W=3, L=153, N=40 µm. The resistive dominant regions (Rs) were

distinguished, according to the conductivity of the solution. In addition, the high (h)

and low (l) cut-off frequencies can also be defined.

I designed the electrodes with two different configurations: (W=3, S=2, L=153)

µm, N=40 for the first-configuration and (W=5, S=10, L=138) µm, N=14 for the

second. Experiments were planned to optimize the electrode with the needle width

limited to 200 μm. The number of fingers was changed by adjusting the width and

spacing of the electrodes. The cell constant of each electrode was calculated to be

2.562 /cm and 12.967 /cm, respectively (capacitance method) [13]. Figure 3-13

summarizes the shift in the high and low cut-off frequencies due to electrode-

geometry modification. An upward shift in the resistive-dominant area, and a shift

of the high and low cut-off frequencies to a lower frequency range are observed. The

impedance spectrum, depending upon the concentration, is the largest in the

resistive-dominant region. If this region appears in a narrow or too-high frequency

range, the non-linear phenomenon of the calibration curve in small measuring

instruments is maximized with a low, usable frequency range. From the frequency-

sweeping results, it is confirmed that the Rs window is shifted to a low-frequency

band; hence, a higher saline-solution concentration can be measured. The

nonlinearity of the measured value was also minimized.

46

Figure 3-23. Frequency sweep result; frequency sweep range 2 Hz to 2 MHz, electrode geometry (W5S10, W3S2)

47

3.4 SU-8 support structure

3.4.1 The interference between plant tissue and electrodes

As the sensor developed in this study is of the insertion type, interference between

the plant and measuring electrode may occur. Therefore, it is critical to study the

distribution of the electric field around the electrodes and the effect of structures such

as the plant tissues. The application of a voltage between the electrodes results in an

electrical field between them, in a conductive solution. The planar interdigitated

electrode design adopted in this study dominates the field phenomenon due to the

fringe effect. When inserted into a plant, the tissue may contact or come close to the

electrode, causing an increase in impedance and noise. I performed simulations using

commercial tool (COMSOL multiphysics) to analyze the electric field generated

between the electrodes and design a suitable structure, which is capable of avoiding

such phenomena, based on the simulation results. The cross section of the

microneedle sensor was reproduced in two-dimension and field analysis was

performed from the electrode in the vertical direction, for determining the influence

range of the field. SU-8 negative photoresist was used as the electrode protection

material. This material has excellent chemical and thermal properties and most

importantly, a high aspect ratio to cover the electric-field range. Figure 3-14 presents

the simulation results of the electrical potential (V) around the two-electrode system.

The fabricated SU-8 support structure on microneedle is shown in Figure 3-15. I

tested the SU-8-structure applicability by mimicking the internal environment of the

plant using hydrogel, which is porous and can absorb the saline solution to create an

environment similar to the interior of the plant. Microneedle sensors with and

48

without the SU-8 structure were inserted into the hydrogel structure to confirm

whether the measurement impedance was affected.

Figure 3-14. COMSOL-multiphysics electrical potential (V) simulation study for planar

electrode

Figure 3-15. Patterned SU-8 dry film on microneedle sensor

49

3.4.2 Sample fabrication and materials

I fabricated a micro scale sensor using a semiconductor etching and micro-

patterning process. Four-inch wafer (p-type, double luster, boron doped, 1-10 ohm)

was used. First electrical passivation layer (silicon oxide, 1 μm) was deposited with

the tetraethyl orthosilicate (TEOS) process (AMK, P-5000). Electrode patterning

was performed using a lift off process using DNR (L300) photoresist as a mask. The

interdigitated electrode patterning was done by lift off process. Negative photoresist

was used as a mask, titanium 300 Å and Au 3000 Å thin film was deposited by

electron beam evaporator (Maestech, ZZS550-2/D). Second silicon oxide

passivation layer (1 μm) was deposited by plasma enhanced chemical vapor

deposition (PECVD) (Oxford instruments, PlasmaPro System100). The top silicon

oxide layer was partially removed by an inductive coupled plasma (ICP) dry etcher

(Oxford instruments, PlasmaPro System100 Cobra) to open the measuring electrodes

and contact pads. SU-8 support resin (SUEX 100) was pattered with the lamination

method (GMP, 655RR). Backside Deep Reactive Ion etching process (PLASMA-

THERM, SLR-770-10R-B) was used to generate thin micro scale needle structure.

This process defined the silicon sensor shape and thickness of the microneedle. High

selectivity of DRIE was achieved using aluminum thin film as a mask layer.

Aluminum mask (5000A) was deposited by sputtering method (sorona, SRN 120).

The remaining aluminum mask layer and organic compound were removed by wet

etching and sonication cleaning. Phase angle measurement was used to

unequivocally investigate the breakdown property compared with visual observation.

Phase angle was monitored by a LCR meter (Agilent E4980A) that can measure

50

impedance values of the whole system.

Figure 3-16. SU-8 applied microneedle fabrication process

51

3.4.3 Simulation and experiment results

COMSOL multiphysics simulation was performed with geometry parameters of

W=5 µm and S=10 µm. The electrical field strength was measured from the

electrode-top along the y-axis, up to 50 µm, as shown in Figure 3-17; the influence

of the field diminished beyond 26.8 µm, from the electrode. The height of the SU-8

structure was 100 µm, considering the safety factor. To verify the effect of the field,

electrodes with and without the SU-8 structure were compared. When a needle was

inserted into hydrogel filled with an electric-conductivity standard solution

(EUTECH) , the sensor with the SU-8 structure showed no noise, as in Figure 3-18.

The thickness of the SU-8 structure was set based on the simulation results so as to

protect the electric field region generated around the electrodes. Figure 3-19

summarizes the working principle of the SU-8 structure. The SU-8 structure creates

a measurement space that may not be interfered by plant tissue.

Numerical analysis and controlled lab experiments were applied to predict and

solve the measurement interference phenomena. Although the sensor developed in

this study, has several advantages for application to horticultural crops, the

interference between the internal plant tissue and the electrode, which is a

disadvantage due to the insertion, was effectively solved by fabricating a support

structure around the electrode.

52

Figure 3-17. COMSOL multiphysics simulation result; electrical field scan from electrode to y-axis

53

Figure 3-38. Real-time measurement to evaluate effect of SU-8 support structure using hydrogel; normal (unprotected) electrode test result (graph above), SU-8 applied electrode test result (graph below)

Figure 3-49. Conceptual diagram of SU-8 supported electrode and unprotected electrode; unprotected electrode (left), protected electrode (right)

54

3.5 Conclusion

In this chapter, I have described the process of an electrical conductivity sensor

that can be inserted into a plant stem. First, several types of silicon micro needles

were made for plant applicability and design optimization. An aluminum mask was

selected to fabricate silicon microneedles and a process was developed to control the

thickness of the needle through two steps of front and back dry etching. The

fabricated needle was applied to the plant stem and a support needle was added for

needle stability. In order to understand the frequency characteristics of the sensor,

measurement and analysis were carried out using NaCl solution. Two electrodes with

different cell constants were evaluated for electrode design optimization. As a result,

the electrode with higher cell constant (W=5, S=10, L=138, N=14) was found

suitable for measuring a higher concentration of electrolyte. However, in order to

widen the measurement range, it is necessary to maximize the active electrode area.

References to this process are covered in more depth in chapter 6. Simulation

analysis tool was introduced to eliminate the obstacles that may occur in the

measurement, and the SU-8 structure was applied to the sensor.

55

3.6 References

[1] Vetter, R. J., Williams, J. C., Hetke, J. F., Nunamaker, E. A., & Kipke, D. R.

(2004). Chronic neural recording using silicon-substrate microelectrode arrays

implanted in cerebral cortex. IEEE transactions on biomedical engineering, 51(6),

896-904.

[2] McAllister, D. V., Allen, M. G., & Prausnitz, M. R. (2000). Microfabricated

microneedles for gene and drug delivery. Annual Review of Biomedical Engineering,

2(1), 289-313.

[3] Fernández, L. J., Altuna, A., Tijero, M., Gabriel, G., Villa, R., Rodríguez, M. J., ...

& Blanco, F. J. (2009). Study of functional viability of SU-8-based microneedles for

neural applications. Journal of Micromechanics and Microengineering, 19(2),

025007.

[4] Zimmermann, S., Fienbork, D., Stoeber, B., Flounders, A. W., & Liepmann, D.

(2003). A microneedle-based glucose monitor: Fabricated on a wafer-level using in-

device enzyme immobilization. In TRANSDUCERS, Solid-State Sensors, Actuators

and Microsystems, 12th International Conference, 1, 99-102.

[5] Windmiller, J. R., Valdés-Ramírez, G., Zhou, N., Zhou, M., Miller, P. R., Jin,

C., ... & Wang, J. (2011). Bicomponent Microneedle Array Biosensor for Minimally-

Invasive Glutamate Monitoring. Electroanalysis, 23(10), 2302-2309.

[6] Amaral, J., Pinto, V., Costa, T., Gaspar, J., Ferreira, R., Paz, E., ... & Freitas, P. P.

(2013). Integration of TMR sensors in silicon microneedles for magnetic

measurements of neurons. IEEE Transactions on Magnetics, 49(7), 3512-3515.

56

[7] Kitano, M., Hamakoga, M., Yokomakura, F., & Eguchi, H. (1996). Interactive

dynamics of fruit and stem growth in tomato plants as affected by root water

condition. I. Expansion and contraction of fruit and stem. Biotronics, 25, 67-75.

[8] De Swaef, T., & Steppe, K. (2010). Linking stem diameter variations to sap flow,

turgor and water potential in tomato. Functional Plant Biology, 37(5), 429-438.

[9] De Swaef, T., Driever, S. M., Van Meulebroek, L., Vanhaecke, L., Marcelis, L.

F., & Steppe, K. (2012). Understanding the effect of carbon status on stem diameter

variations. Annals of botany, 111(1), 31-46.

[10] Olle, M., & Williams, I. (2015). The influence of effective microorganisms on

the growth and nitrate content of vegetable transplants. Journal of Advanced

Agricultural Technologies, 2(1).

[11] Izumi, H., & Aoyagi, S. (2007). Novel fabrication method for long silicon

microneedles with three-dimensional sharp tips and complicated shank shapes by

isotropic dry etching. IEEJ Transactions on Electrical and Electronic Engineering,

2(3), 328-334.

[12] Ganji, B. A., & Majlis, B. Y. (2006). Deep trenches in silicon structure using

DRIE method with aluminum as an etching mask. In Semiconductor Electronics,

2006. ICSE'06. IEEE International Conference. 41-47.

[13] Jeon, E., Choi, S., Yeo, K. H., Park, K. S., Rathod, M. L., & Lee, J. (2017).

Development of electrical conductivity measurement technology for key plant

physiological information using microneedle sensor. Journal of Micromechanics and

Microengineering, 27(8), 085009.

57

Chapter 4. Measurement system

4.1 Electrical conductivity measurement system overview

EC measurement is based on the impedance measurement of saline solution in

contact with the electrodes. The measured impedance is converted to electrical

conductivity based on standard solution (EUTECH, Singapore) calibration.

Impedance measurement modules and communication devices have been

miniaturized and connected to micro needle sensors in order to apply to real plants.

I used an impedance converter chip (AD5933, Analog Device) to measure the

impedance between two electrodes on the surface of the microneedle. The AD5933

is a high accurate impedance conversion system consisting of frequency generator

and a temperature measurement sensor and has ability to convert analog to digital

signals. Figure 4-1 shows block diagram and simplified chip configuration of

AD5933 Analog to Digital Convertor (ADC) chip.

Figure 4-1. AD5933 12-bit impedance converter; functional block diagram and chip configuration [1]

58

Figure 4-2 shows the schematic of packaging of the microneedle and other

components. I programmed the impedance converter chip with I2C (inter-integrated

circuit) communication protocol using a microcontroller (Arduino Uno, company).

I set the amplitude and the frequency of the measurement signal at 0.4 mVp-p and

10 kHz, respectively. The chip generates the measurement signal from the output

pins, and measures the amplitude of the current through the input pins. The current

is changed to voltage using the current-to-voltage amplifier embedded in the chip,

and convert to digital values through the digital to analog converter (DAC). The

digital data were processed via the microcontroller and transmitted to the PC using

the Bluetooth wireless protocol. The digital values were finally combined with the

EC values in the PC for real-time monitoring

Figure 4-2. Microneedle probe electrical conductivity measurement system integrated with AD5933; (a) microneedle chip integration with PCB using wire bonding (b) microneedle impedance analyzer package

In case of plant measurement, the ground effect is a critical issue as plant is

grounded through its roots. This causes current leakage that affects the AD5933

current measurement. In addition, AD5933 chips interfere with each other because

59

they share the same medium in this case, the plant xylem. I solved this issue by

isolating every AD5933 chip using a relay. I connected only one AD5933 during

each measurement. The entire system of electrical conductivity measurement sensor

is as shown in Figure 4-3

Figure 4-3. Schematic of microneedle measurement device and details of relay system for AD5933 isolation

60

4.2 Device operation and calibration

This section describes the operation and calibration procedure of the system based

on the impedance converter chip. This process was based on an evaluation board

with AD5933 and a microcontroller (Arduino) in Figure 4-4. Simplified

methodology applied to measurement package.

2 2Magnitude R I= + (4-1)

1ImpedanceAdmittanceGain factor

Code magnitude

= =

(4-2)

1Impedance = Gain Factor Magnitude×

(4-3)

The important factors in the process of calculating the impedance in the module are

magnitude, gain factor, and calibration resistor. R and I shown in Eq (4-1) are real

number and imaginary number, respectively. The calculated value is multiplied by

the gain factor and expressed as an impedance in Eq (4-3). The equation for obtaining

the gain factor is shown in Eq (4-2). The calibration resistor is used to calculate the

gain value. The stabilization range of the measured value differs depending on the

calibration resistance set. Figure 4-5 shows that the signal stabilization range is set

according to the resistance setting. It can be seen that stability is poor in the region

below the resistance of 39 kohm used in the calibration, and stable signal in the high

61

region. The process of calibrating the sensor module is depicted in Figure 4-6.

Figure 4-4. System operation schematic with AD5933 evaluation board and microcontroller (Arduino)

62

Figure 4-5. Magnitude signal variation via calibration resistance change in AD5933 system

Figure 4-6. System module calibration sequence: 1) pick target resistor, 2) measure gain and magnitude with various known resistors, 3) calculate the noise of impedance, 4) define target range

63

4.3 system evaluation

4.3.1 Signal to Noise Ratio (SNR) of device

The microneedle electrical conductivity (MEC) measurement circuit was designed

and evaluated by SNR. The microcontroller was Arduino bluno (ARDUINO).

Switched-Mode Power Supply (SMPS) was used to apply a uniform voltage to the

sensor. An ADuM4160 digital isolator (Analog Device) was used to isolate the noise

from the source supplied by the power supply. The AD5933 impedance converter

(Analog Device) 12 bits was used for the ADC. Entire experiment set up is shown in

Figure 4-7.

The performance of the fabricated MEC noise reduction measurement system was

evaluated. In the evaluation method, a precision resistor was connected to the

measurement module, the temperature was kept constant, and the signal value was

recorded. The signal-to-noise ratio of the sensor was determined using the average

value and the noise value of 800 digital signal values recorded at this time. First, the

SNR of the system was measured as 91.9 (SNR-1 = 0.0108819) using the SMPS alone

in the power supply without applying the noise reduction factor in Figure 4-8. In this

case, the measurement itself is possible, but when applied to plants, it is impossible

to measure accurately when considering the additional noise. The signal-to-noise

ratio (SNR) of the system with a noise reduction system was measured at 1551.7

(SNR-1 = 0.0006444) in Figure 4-9.

64

Figure 4-7. Measurement system noise evaluation setup

Figure 4-8. Measurement system noise evaluation with high precision resistor (without ground isolation system)

65

Figure 4-9. Measurement system noise evaluation with high precision resistor (with ground isolation system)

4.3.2 Impedance characteristics (AD5933 integrated sensor)

The AD5933 chip has an operating frequency range of 2–100 kHz. Hence, it is

important to find a frequency band suitable for the system within the allowable range.

For the impedance measurement, I used six standard solutions having conductivity

in the range 0.084 mS/cm to 12.800 mS/cm. During the experiment, the frequency

was increased from 2 kHz to 100 kHz in increments of 2 kHz between successive

frequencies. Figure 4-10 shows the results of the frequency spectroscopy conducted

using the AD5933 measurement system. A standard solution higher than 0.500

mS/cm within the driving frequency enters the electric double layer capacitance

66

dominant region ( EDLC ). From an initial frequency of 2 kHz, an impedance gradient

is generated according to the standard solution electrical conductivity. The electrode

fabricated in this study was directly exposed to the solution. Exposing the electrode

to the conductive solution results in formation of an electric double layer on the

surface of the electrode and the layer thickness changes according to the

concentration of the solution. Thus, even at very low frequencies, an impedance

gradient was created for the standard solution. I measured the impedance value

according to the solution conductivity in the representative frequency band to find

the optimum driving frequency in the measurable frequency range.

Table 1. shows the measured impedance values in each frequency domain used to

determine the driving frequency. Analysis of the overall measurements reveals that

the measured values in the high concentration range have nonlinear characteristics.

The impedance is saturated in the high concentration region due to the polarization

of the electrode surface. A decrease in impedance with increase in frequency can be

observed in Table 1. I also encountered a decrease in impedance ∆Z (∆Z = Z5.00 mS/cm

- Z12.800 mS/cm) at high frequency. Comparing the three frequencies, the most suitable

value was measured in the 10 kHz range. At 10 kHz, the ∆Z value was the largest.

Taking into account the 0.1–10 mS/cm EC measurement objective, I selected an

operating frequency of 10 kHz.

67

Figure 4-10. Frequency sweep test result; impedance measurement module integrated with AD5933 impedance analyzer chip

Table 4-1. Frequency sweeping results with an AD5933 and measurement circuit Impedance measurement (AD5933)

Frequency Standard solution (mS/cm) 0.084 0.500 1.413 2.764 5.000 12.800

10 kHz 160.52

kΩ 79.49 kΩ 63.84 kΩ 56.17 kΩ 51.90 kΩ 46.99 kΩ

50 kHz 97.92 kΩ 30.87 kΩ 19.81 kΩ 16.65 kΩ 15.02 kΩ 13.69 kΩ

100 kHz 69.91 kΩ 24.29 kΩ 14.94 kΩ 12.77 kΩ 11.53 kΩ 10.36 kΩ

68

4.3.3 Real-time impedance monitoring

The impedance change with the addition of standard solution in the chamber in real

time result is shown in this chapter. Real-time monitoring is conducted using

AD5933 based measurement system. Three different conductivity solutions were

formed by adding 200 µl, 400 µl, and 600 µl standard solutions with conductivity of

12.800 mS/cm each to 20 mL D.I water. Addition of these solutions to the chamber

resulted in immediate change in impedance, as depicted in Figure 4-11. The

highlighted area represents the non-specific point resulting from the diffusion of the

locally added saline solution. It was confirmed that the signal stabilized immediately

after stirring the solution. In addition, horticultural crops such as tomatoes exhibited

a steady flow during the daytime or nighttime through xylem or phloem [2]. Thus,

diffusion noise was expected to be small, as shown in the graph in Figure 4-11.

Another phenomenon seen in the results is a nonlinear tendency in which the change

in impedance due to concentration changes becomes smaller as the concentration

increases. By setting the resistance of the calibration resistor around the impedance

measured in a high concentration solution, nonlinear compensation can be achieved

with high accuracy, even at low variations. When the concentration of the solution

changes from 0.397 to 0.762 mS/cm, the signal to noise ratio was 24.68.

69

Figure 4-11. Real-time monitoring result of concentration change due to additional supply of concentrated solution

70

4.3.4 Standard solution calibration

Calibration was performed to convert impedance to electrical conductivity.

Empirical methods were utilized to calibrate the measurement device. In the

calibration process, the impedance of the standard solution was first measured to

generate a calibration index. Next, the impedance vs EC calibration curve was

generated using linear curve fitting, to give the result shown in Figure 4-12.

Experimental Data (number of experiments = three) was tabulated as shown in Table

2. As can be seen in Figure 4-13, owing to the characteristics of the impedance

measurement system, a low variation was observed in the high concentration region

(1.413–12.880 mS/cm), whereas a high variation was observed in the low

concentration region (0.084–5.000 mS/cm). Contribution to the overall impedance

is dominated by the EDL capacitance. At low concentrations the thickness of EDL is

larger than high concentrations. Such variation in the EDL thickness is exponentially

correlated with the concentrations, leading to highly nonlinear behavior of the

calibration. In addition, as the measurement involved high concentration EC solution,

there exists a possibility of contamination on the electrode surface, resulting in

impedance shifting.

71

Figure 4-12. Microneedle electrical conductivity sensor calibration using various concentration of standard solution (0.084 ~ 12.880 mS/cm, linear fitting)

72

Figure 4-13. Three sets of calibration curves; Three different microneedle sensor evaluated by standard solution

Table 4-2. Calibration results of standard solution with an AD5933 measurement

circuit

Impedance measurement (AD5933)

Needle Standard solution (mS/cm)

0.084 0.500 1.413 2.764 5.000 7.940 12.800

Sensor_1 (kΩ) 167.28 98.43 66.36 59.69 52.05 42.10 40.33

Sensor_2 (kΩ) 187.29 91.76 67.52 57.05 51.78 45.73 41.13

Sensor_3 (kΩ) 185.34 88.73 66.15 57.83 51.93 46.36 42.26

Standard

deviation 11.03 4.96 0.74 1.36 0.14 2.30 0.97

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4.3.5 Temperature effect evaluation

In this study, an experiment was conducted to confirm the effect of temperature

on impedance. A microneedle was inserted into the teflon channel to mimic the plant

vesicular bundle. Figure 4-14 shows the experiment set-up and the result graph

respectively. As a result, it was confirmed that the impedance measurement value

changed by 0.614 % as the temperature changed by 1 degree in Figure 4-15.

Introducing temperature gradient of 2.73 °C results in 1.68 % change in impedance.

Considering that the daily variation of relative internal temperature of the plant, the

temperature effect on the measurement is insignificant. The results for plant internal

temperature are discussed in chapter 5. Nevertheless, in order to improve accuracy,

I am looking for a way to compensate for temperature.

Figure 4-14. Test setup for temperature effect on microneedle sensor. A Teflon tube was used to create a plant vesicular bundle like environment

74

Figure 4-15. Test results for temperature effect on sensor

75

4.3.6 Array sensor evaluation

In this study, microneedle chips fabricated using semiconductor processes include

array of electrodes. A single or multiple electrodes are operated depending on the

purpose. In real-time experiment, single electrode measurement was performed.

However, multiplex sensing platform was designed due to morphological aspect for

plant stem. Two different types of plant were concerned. Monocotyledon and

dicotyledon have different internal stem structures. In dicotyledon plants, the

cambium is arranged in a ring shape on the edge of the stem, and the xylem and

phloem are located inside and outside of the ring, respectively. In contrast, in case of

monocotyledon plants, there is no cambium, and xylem and phloem are scattered

throughout the stem. Horticultural crops such as tomatoes, cucumbers and paprika,

which are the target plants in this study, have dicotyledon structures. Thus, the

electrode array formed along the length of the needle can increase the probability of

an electrode placed in the xylem or a phloem. Furthermore, the position of the needle

insertion can be determined through signal analysis.

The microneedle array sensor includes three impedance measurement modules

and one microneedle chip. All impedance measurement modules are integrated and

controlled using a digital mux. Impedance module and microneedle chip are

combined into one package using 3D printed structure. Figure 4-16 shows the

components of a microneedle array system.

Experiments were conducted to evaluate array sensor performance. The

experiment was carried out on tomato plants previously mentioned in this study.

Tomato is a dicotyledon plant that offers easy access to xylem and phloem.

76

Experiments were conducted in both laboratory and greenhouse settings as shown in

Figure 4-17. The plants used were tomatoes grown in greenhouse, and the tomatoes

used in the laboratory were transferred to the laboratory and treated with 1.4 mS/cm

nutrient solution and halogen lamp. The growth conditions of the tomato in the

greenhouse are explained in detail in chapter 5.4.2.

Figure 4-16. Illustration of microneedle array sensor schematics and sensor package

77

Figure 4-17. The experiment setup and details of microneedle: (a) experiment setup at lab scale approach, (b) experiment setup and test period for greenhouse, (c) the microneedle sensor parametric details for needle insertion

78

From the experimental results in Figure 4-18 and Figure 4-19, I could find three

facts. The first one is about the current sensor design. When applied to a stem

diameter of 6.8 mm, the sensor No. 1 cannot be placed in xylem or phloem. By

cutting and staining the stem, I measured the width of the structure including the

water tube, body tube and epidermis to be about 2 mm. Second, the electrical

conductivity difference between day and night was confirmed. At night, root pressure

is not caused by evaporation, but only absorption by osmosis occurs. In order to

absorb moisture, the internal ion concentration must be higher than the external ion

concentration. Therefore the electric conductivity level is higher than the daytime.

Third, there is a difference in measured values depending on the electrode position.

By applying array sensor, it is possible to measure changes according to internal

structure of plants.

Figure 4-18. Microneedle array sensor test result (at night 20:54 ~ 21:38)

79

Figure 4-19. Microneedle array sensor test result (at day 13:58 ~ 15:07)

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4.4 Conclusion

I designed an electrical conductivity measurement package for use in plants and

evaluated its operating performance at laboratory scale. The measurement system

with AD5933 was designed and calibrated. The calibration resistor was set to specific

gain. There is variation among each AD5933 chip, but the obtained gain value did

not exceed ±1e-10 value of 128.22e-10. Noise reduction techniques have been

applied to the measurement module and the signal-to-noise ratio of the

measurements has been found to be 1551.7 (SNR-1 = 0.0006444). Using the

completed calibration module, frequency sweep test, microneedle calibration,

temperature effect, and real-time monitoring test were performed.

Figure 4-20. Real-time monitoring sensor system with microneedle sensor

81

As a result of module evaluation, the largest variation window of the impedance

between the concentration within the target measurement range was found at the

measurement frequency of 10 kHz. Therefore, the operating frequency was fixed at

10 kHz. Immediate response to real-time concentration changes was identified.

Noise due to diffusion was found but was removed as the concentration gradient

stabilized.

The effect on temperature was measured with 0.614% impedance change for 1

degree Celsius. Figure 4-20 shows final measurement setup. By evaluating the

fabricated microneedle sensor package, the operation possibility of the measurement

module was verified. In addition, a microneedle array sensor was fabricated and

evaluated. Through the array sensor measurement, I confirmed the signal difference

according to internal structure and position of plant stem. In addition, daytime and

night time signal differences were found. Experiments have further strengthened the

need for array type sensors and deepened understanding of plant internal signals.

4.5 References

[1]http://www.analog.com/media/en/technical-documentation/data-

sheets/AD5933.pdf

[2] Peuke, A. D., Rokitta, M., Zimmermann, U., Schreiber, L., & Haase, A. (2001).

Simultaneous measurement of water flow velocity and solute transport in xylem and

phloem of adult plants of Ricinus communis over a daily time course by nuclear

magnetic resonance spectrometry. Plant, Cell & Environment, 24(5), 491-503.

82

Chapter 5. Real-time monitoring measurement for

electrical conductivity inside of cucumber stem

A microneedle sensor was developed to measure the electrical conductivity,

important information inside the plant. The electrodes were selected according to the

requirements of the measurement environment, application target and evaluated

using an LCR meter. Silicon-based needles were fabricated and integrated into the

developed measurement package, making them applicable to horticultural plants.

Experiments were conducted on cucumbers at Ilsanseo-gu, Goyang-si, Gyeonggi-

do, Republic of Korea for application involving actual plant monitoring. The real-

time monitoring for cucumber plants were carried out for 48 hours and specific

signals were obtained through four sensors. In addition, studies on the defense

mechanisms of plants have been verified and reasonable solution was suggested.

5.1 Cucumber cultivation for experiment

The experiments were performed in a greenhouse at Beopgot-dong, Ilsanseo-gu,

Goyang-si, Gyeonggi-do in the Republic of Korea, located at a latitude of

37°39'56.8" N and a longitude of 126°42'54.0" E in the Figure 5-1. Cucumber seeds

(wonnongseed, Cucumis sativus L) were sown in 40 hole-trays on October 2, 2016.

Before the transplantation, the slabs were flushed with nutrient solution at 5.5–5.8

pH approximately and an electrical conductivity of 1.8 mS/cm. The compositions of

the nutrient solution were as follows: Solution A = calcium nitrate (Ca(NO3)2,

potassium nitrate (KNO3), and Iron chelate (Fe-EDTA (ethylene

83

diaminetetraacetate); Solution B = potassium nitrate (KNO3), magnesium sulfate

(MgSO4–7H2O), potassium phosphate monobasic (KH2PO4), and ammonium

phosphate monobasic (NH4H2PO4); micronutrient = boric acid (H3BO3), copper

sulfate (CuSO4), zinc sulfate (ZnSO4), Manganese sulfate (MnSO4), and sodium

molybdate (Na2MoO4). The cucumber seedlings were transplanted on November 11,

2016 to a commercial slab (Seo Won Co., Cocomix, 15x10x100cm). The irrigation

of the nutrient solution was adjusted according to the amount of solar radiation, using

a drip irrigation system (Priva Maximizer) after transplantation. The electrical

conductivity of the nutrient solution was maintained at 2.3 mS/cm and the pH was

maintained at 5.5–5.8. The humidity of the green house was 70– 80% during the day

and 90–95 % at night. The room-temperature was controlled in four steps: Sunrise

to afternoon (25–28° C), afternoon to sunset (23–25° C), 3 h after sunset (15–16° C),

and until sunrise (13° C). The real-time environmental conditions such as the solar

radiation (W/m2), temperature (°C), relative humidity (RH %), and the carbon

dioxide dose were monitored using built-in sensors (Priva Maximizer) in the

greenhouse.

84

Figure 5-1. Greenhouse cucumber cultivation and real-time monitoring experiment

5.2 Relative temperature in the plant stem

5.2.1 Relative temperature measurement methodology

A miniature resistance thermometer was inserted into the plant stem to measure

the temperature change inside the plant. The measurement method used the principle

of a general RTD (Resistance Temperature Detector) to calculate the temperature

from the resistance change of the conductor. A resistance thermometer was fabricated

by patterning gold on a silicon substrate with a constant resistance and processing it

into specimens. The thermometer measures resistance in an ohmmeter and stores

data converted from PC to temperature.

85

5.2.2 Relative temperature in cucumber stem

Sensors using electrochemical measurement methods are sensitive to temperature

changes. In electrical conductivity measurements, relatively high conductivity

values are measured at high temperatures and relatively low values at low

temperatures. The sap inside the plant migrates to the phloem and xylem, and is

affected by various mechanisms. The plant interior is protected by the cortex and the

epidermis; hence, the temperature of the external greenhouse and the temperature of

the internal stem of the plant may be different. It is necessary to analyze changes in

the plant stem internal temperature to make accurate electrical conductivity

measurements. Figure 6-3. shows the measured relative temperature change of the

cucumber stem. The graph shows that the relative temperature change within the

plant stem was up to 2.90 °C. In a greenhouse, the ambient temperature changes

between 15.06 °C and 29.75 °C. There is relatively less change in the internal

temperature compared to the outside temperature change of the greenhouse.

Controlled experiments conformed that impedance changed by 0.614 %

corresponding to 1 °C change in temperature. Temperature change of 2.90 °C within

the plant stem results in 1.78 % impedance change, which is minor contribution to

overall measurement. The details for relative temperature measurement in plant stem

are provided in supplementary material.

86

Figure 5-2. Temperature change in and outside of cucumber stem: relative temperature (straight line), ambient temperature monitored by temperature sensor integrated with AD5933 (dotted line)

5.2.3 Correlation between external temperature and relative temperature

The difference between the inside temperature and the outside temperature of the

plant stem is large. As mentioned in chapter 5.2.1, the sensor temperature effect is

negligible. However, the analysis was carried out to understand the correlation

between internal and external temperature. The internal and external temperature

changes measured at the same time are plotted in a decentralized graph. As shown in

Figure 5-3, the internal and external temperature variations have a linear tendency.

This result suggests the possibility of converting the external temperature to the

corresponding internal temperature.

87

Figure 5-3. Correlation between internal relative temperature and external relative temperature. The linear correlation was observed

88

5.3 Plants defense mechanism

The microneedle sensor inserts a needle into the stem to measure plant internal

information. The act of penetrating the stem can be perceived as a wound or invasion

of the plant. Therefore, the defense mechanisms of plants were examined.

The phloem is an important component to transport nutrients. Inside the tube,

important elements such as starch, ions, and proteins are delivered to each site for

plant growth. If nutrients of the plants are exuded by external factors, callose and p-

protein are activated. Callose blocks the pore of sieve plate to prevent fluid leakage

[1-2]. The P-protein is released to the wound site at the same time as the sap flows

out. The solution comes into contact with oxygen and oxidizes to a gel form [3]. The

wounded area is completely blocked by the gel, which can prevent internal fluid loss.

Figure 5-4. Schematic of the plant defense mechanism for wound

89

Figure 5-4 illustrates the defense mechanism of the plant after a wound. When a

needle is inserted into a plant, a wound is created as shown in 5-5. The wound healing

process initiated shortly by the plants thereby make long term measurements

challenging. Figure 5-6 shows that the measuring electrode of the micro-needle is

coated by the gel thereby making real time measurements difficult. In order to

prevent such a phenomenon, a sealing material is applied to prevent the sap from

being exposed to the outside as shown in 5-7. This method is similar to secreting

saliva when an insect takes up the nutrients of the phloem [4-6]. The sealing process

was applied to obtain real - time measurement data for two days.

Figure 5-5. Schematic of the plant defense mechanism effect to needle insertion

90

Figure 5-6. Schematic of the slime coated needle (left) and interdigitated electrode (right)

Figure 5-7. Schematic of the needle installation with sealing technique

91

5.4 Cucumber electrical conductivity monitoring

5.4.1 Electrical conductivity of plant sap

The electrical conductivity of the plant was measured by inserting a micro needle

sensor into the vesicular bundle of plant stem. The vesicular bundle is composed of

xylem and phloem. The xylem carries moisture and various minerals, and the phloem

carries nutrients necessary for growth. Plants have a pattern according to the day and

night cycle. In the case of xylem, relatively low electrical conductivity is obtained

due to the influence of water absorbed from the roots at daytime, while at night,

water absorption takes a relatively high value due to the osmotic action. In the case

of phloem, it exhibits an opposite periodicity. The phloem shows a relatively high

electrical conductivity level during the day time than night time.

5.4.2 Real-time change of electrical conductivity in plant stem

It is confirmed that the performance of the sensor is reasonable based on the

laboratory operations. However, the result in an ideal environment is not only

convincing but also informing the possibility of operation. Therefore, a cucumber

plant grown in a greenhouse environment was prepared for field test. Figure 5-8

shows the cucumber used in the experiment. The experiments were performed in a

greenhouse at Bongcheon-dong, Gwanak-gu, Seoul in the Republic of Korea, located

at a latitude of 37°47'18.93" N and a longitude of 126°95'81.80" E. The nursery plant

(Takii, Super Dotaerang) were transfered into slab (Grodan, Rockwool) on

November 27, 2017. Greenhouse internal temperature was controlled between 13.5 °

92

C to maximum 25 ° C. The electrical conductivity of the nutrient solution (Standard

solution from protected horticulture research station in Korea) was maintained at 2.0

mS/cm and the pH was maintained at 6.5. The real-time environmental conditions

such as the solar radiation (W/m2), temperature (°C), relative humidity (RH %), and

the carbon dioxide dose were monitored using built-in systems (Shinhan A-tech,

Aqua-M and SH-3000) in the greenhouse.

In order to confirm the change of electrical conductivity in the plant, 700 mL tap

water was applied. The experiment started at noon and proceeded for about 2 hours

and 20 minutes. The sensor value was about 0.6 ~ 0.8 mS/cm at noon. In a few

minutes, the value of electric conductivity measured after the application of 700 mL

of tap water sharply decreased. These results occurred because the concentration of

electrical conductivity in the slab was rapidly lowered and the ion concentration of

water absorbed in the root was also lowered. From these results, it was confirmed

that the internal conductivity of the plant stem could be measured using the

developed sensor inturn observing the change of internal ion concentration to the

external environment.

93

Figure 5-8. Cucumber plants grown in a greenhouse environment for experiment. Cucumbers used in the experiment are indicated by black arrows

Figure 5-9. The real-time monitoring electrical conductivity measurement result; immediate observation of changes in electrical conductivity value reflecting external environmental changes

94

5.4.3 Cucumber monitoring data

Figure 5-10, 5-11, 5-12, 5-13 displays the data measured for two days, after the

installation of the sensor on the cucumber stem. Four microneedle sensors were

installed on different cucumber stems (n=2) for data collection. Two microneedle

sensor were installed near the root and the other two were placed middle of entire

cucumber plant.

Figure 5-10. Microneedle sensor inserted in cucumber stem

95

The AD5933 chip, which measures the impedance, has a built-in temperature

sensor to measure the ambient temperature. However, as mentioned in previous

research reports, the temperature change inside the plant is negligible compared to

the outside temperature change. In addition, the temperature effect of the

microneedle sensor was 0.614% per degree, which is a minor contribution to the

overall measurement [7].

Figure 5-11. The real-time monitoring electrical conductivity measurement result; sensor 1 (mS/cm) at ambient temperature (degree Celcius)

Figure 5-10 shows installed microneedle sensor. The sensor was consisted of

microneedle, AD5933 module and microcontroller. The measured impedance signal

is connected to the microcontroller through the wire and transmitted to the computer

using wireless communication. A sealing material was applied to avoid measurement

failure due to plant defense mechanisms.

96

Three findings can be identified from the real-time electrical conductivity

monitoring of cucumber plants. First, the level of the sap electrical conductivity

measured for cucumber. The internal components of the plant sap approximate the

components of the displaced soil solution. In the past, this has been confirmed using

the bleeding method [8]. The electrical conductivity of cucumber, in this experiment,

was maintained at 2.3 mS/cm. Considering the value of the maintained electrical

conductivity, the measured electrical conductivity value was found to be reasonable.

Figure 5-12. The real-time monitoring electrical conductivity measurement result; sensor 2 (mS/cm) at ambient temperature (degree Celcius)

Second, daily cycle changes were observed. The electrical conductivity at

night is relatively higher than at daytime. Plants have different day and night ion

concentrations. The concentrations of the major plant ions (K, Ca, Mg, P, Na etc.)

tend to decrease at daytime whereas an increase is observed at night [9]. The

electrical conductivity results of the sensor indicate this cyclic nature. Third, the

97

xylem can be specifically accessed through the measured signal pattern. Both sensor

data exhibit different electrical-conductivity levels and behavior. In the case of

sensor 1,3 and sensor 4, the periodicity and the change can be observed clearly.

However, sensor 2 exhibited a reasonable electrical conductivity value but no

apparent ion change was observed. By analyzing the measured signal pattern using

the ion-change characteristics of the xylem (Sensor 1, 4) and phloem (sensor 3), it is

possible to verify the zone for needle insertion. The overall sensor measurement

signal exhibited high noise in the high-electrical-conductivity region (over 4 mS/cm),

in particular. This is caused by the nonlinear characteristics of the calibration curve.

Figure 5-13. The real-time monitoring electrical conductivity measurement result; sensor 3 (mS/cm) at ambient temperature (degree Celcius)

98

Figure 5-14. The real-time monitoring electrical conductivity measurement result; sensor 4 (mS/cm) at ambient temperature (degree Celcius)

99

5.5 Conclusion

This study analyzed the performance of a microneedle device with an embedded

impedance system and wireless communication package. The micro needle sensor

was directly inserted into the plant to confirm the real-time measurement capability.

In addition, I confirmed the possibility of periodicity of plant and selective

measurements through two-day measurement. Plants have a defense mechanism

against wounds. Therefore, research was conducted to solve this problem and a

sealing method was proposed. The proposed method works effectively and can

measure up to 17 days.

100

5.6 References

[1] Evert, R. F., & Derr, W. F. (1964). Callose substance in sieve elements. American

Journal of Botany, 552-559.

[2] Xie, B., Wang, X., Zhu, M., Zhang, Z., & Hong, Z. (2011). CalS7 encodes a

callose synthase responsible for callose deposition in the phloem. The Plant

Journal, 65(1), 1-14.

[3] Cronshaw, J., & Esau, K. (1968). P protein in the phloem of Cucurbita. The

Journal of cell biology, 38(1), 25-39.

[4] Will, T., Furch, A. C., & Zimmermann, M. R. (2013). How phloem-feeding

insects face the challenge of phloem-located defenses. Frontiers in Plant Science, 4.

[5] Will, T., & Vilcinskas, A. (2015). The structural sheath protein of aphids is

required for phloem feeding. Insect biochemistry and molecular biology, 57, 34-40.

[6] Gunning, B. E., & Steer, M. W. (1996). Plant cell biology: structure and function.

Jones & Bartlett Learning.

[7] Eunyong, J., Seungyul, C., Kyung-Hwan, Y., Kyoung Sub, P., Mitesh, L. R., &

Junghoon, L. (2017). Development of electrical conductivity measurement

technology for key plant physiological information using microneedle sensor.

Journal of Micromechanics and Microengineering, 27(8), 085009.

[8] Lowry, M. W., & Tabor, P. (1931). Sap for analysis by bleeding corn

plants. Science, 73, 453.

[9] Peuke, A. D., Rokitta, M., Zimmermann, U., Schreiber, L., & Haase, A. (2001).

Simultaneous measurement of water flow velocity and solute transport in xylem and

101

phloem of adult plants of Ricinus communis over a daily time course by nuclear

magnetic resonance spectrometry. Plant, Cell & Environment, 24(5), 491-503.

102

Chapter 6. Optimization and enhancement

6.1 Gold Nano Rod (GNR) array on electrode

6.1.1 Enhanced sensor performance by surface modification

Depending on the type of electrode used and the geometry of the electrode, the

range and application of the conductivity cell was selected. As mentioned in chapter

2, this study uses interdigitated electrodes in consideration of measurability in a wide

measurement range and a closed environment. However, due to the miniaturization

of the entire platform of the sensor, the electrode area becomes extremely small and

a measurement error due to the polarization phenomenon may occur. In addition, the

non-linear phenomenon occurs in the calibration process, and the resolution may be

lowered in the high-density region to increase the noise. To solve this problem and

optimize the measurement range, it is essential to maximize the electrode surface

area.

Nano rod arrays are frequently used to maximize sensor performance through

increased surface area [1-3]. Figure 6-1 shows a GNR array formed on a thin metal

film. Based on image processing, a total number of 109 GNR can be fabricated

within an 1 1m mµ µ× area. The difference in the area between the plane and the

surface on which the GNR is formed is 100 times.

1( )( )low

EDL E

ZC C

ωω

=+

(6-1)

103

The following Eq (6-1) shows the slope of the region where the influence of EDLC

is dominant. Figure 6-2 is a conceptual diagram showing the characteristics of the

electrode according to the frequency change. The EDLC value is proportional to the

electrode area. If the electrode area is maximized, the measurement window can be

widened by lowering the low cut off frequency as shown in Figure. 6-3. It can also

reduce polarization effects. In this experiment, GNR manufacturing process was

applied to MEMS fabrication process. Also, it is confirmed that the frequency range

is broadened by maximizing the electrode area.

Figure 6-1. Increase electrode surface area modifying GNR array on electrode; 109 single GNR fabricated on 1 µm x 1 µm area. Single GNR diameter is 70 nm

104

Figure 6-2. The schematic of frequency region according to electrode configuration

Figure 6-3. The schematic of frequency region changes according to increase electrode surface area (Effect of EDLC modification)

105

6.2 GNR array fabrication and results

6.2.1 GNR array fabrication on electrode

Semiconductor process technology was used to form GNR on the electrodes. A

500 µm thick, 4 inch (100) diameter silicon wafer is used as the substrate. Because

both the front and back sides of silicon are processed, double-sided polishing wafers

are used. A 1µm silicon oxide insulating layer is deposited on the top surface of the

wafer using a Tetraethyl orthosilicate (TEOS) CVD process. On this, mask

patterning is performed using a negative photoresist for the lift off process. Then,

3000Å of gold is deposited by evaporation deposition method.

Figure 6-4. Apparatus for oxidation process: (a) wafer scale processing zig made of PEEK material with strong chemical resistance (b) internal cross-section of equipment (c) elastic electrode to reduce contact resistance and protect sample surface (d) ring electrode

106

The deposited wafer is patterned into a desired electrode shape by lift off using

acetone and sonication. The residue before depositing the secondary insulation layer

is removed by the descum process. An upper insulating film of 1 um is formed by

PECVD. After photoresist masking, ICP etching is performed to create selectively

openings.

The GNR is manufactured using anodized aluminum oxide (AAO) as a frame.

Therefore, an aluminum thin film having a thickness of 2 µm is deposited. The

thickness of the thin film can be changed according to the GNR height to be

manufactured. The prepared wafer is inserted into the device shown in Figure 6-4.

Oxalic acid electrolytes are loaded into the chamber for the oxidation process. The

aluminum thin film is oxidized by the constant voltage method. At this time, a nano

scale hole is formed in a direction perpendicular to the surface. The vertically formed

hole is connected to the gold electrode under the aluminum oxide film. After the

reduction process, gold is electrodeposited and phosphoric acid is used to remove

AAO. The samples fabricated in wafer units were processed into pieces using dicing

equipment.

107

6.2.2 Fabrication results

The sample was processed according to the procedure, followed by Scanning

Electron Microscope (SEM) analysis. Figure 6-5 is a SEM image of GNR formed on

the prepared sample and contact pad (arrow). Gold was used as an electrode-based

and electroplating seed layer. The reason why the electrodes in the figure are red is

due to the light reflected by the formed GNR. The GNR height is measured to be

about 1 µm. The larger the distance between the rods, the closer to red the light

reflects, the narrower the gap, the reflection shifts towards blue [4]. According to the

results of SEM measurement, the diameter of GNR is 70 nm, height is about 1 um,

and the gap is 30 nm. In the sample, except for the measuring electrode and the

contact pad, the remaining portion is covered with a silicon oxide insulating layer.

Thus, selective GNR formation is possible. The degree of GNR formation of the

electrode is not perfect. In the oxidation process, i assume that the GNR cannot be

formed because the current cannot flow to the electrode finger having a relatively

high resistance. The measurement electrode with the GNR and the SEM image are

shown in Figure 6-7. The portion displayed black on the measuring electrode is the

portion where the GNR is formed. It can be confirmed that GNR is formed only in a

region having a relatively wide width.

108

Figure 6-5. Fabricated surface modified GNR sensor; diced GNR sample (left), 12.3 mm x 30 k SEM image (upper right), 12.3 mm x 200 k SEM image (lower right)

109

Figure 6-6. Interdigitated electrode integrated with GNR array; 12.3 x 35.0 k SEM image for GNR (upper), GNR on the interdigitated electrode (lower left), fabricated sensor (lower right); arrow points out the imaged GNR on the chip

110

6.2.3 Test results

The frequency generator and the impedance analyzer used in the experiment were

modules with AD5933. The voltage was 0.4 Vp-p and the frequency was increased

sequentially from 2 Hz to 100 kHz. Four standard solutions were used (standard

solutions: 0.5 mS/cm, 1.413 mS/cm, 2.764 mS/cm, and 5.0 mS/cm) to obtain

concentration specific results.

Figure 6-7. Frequency sweep result for planar interdigitated electrode. The unit of the standard solution is µS/cm

111

Planar electrodes were fabricated to compare the results with the samples with

GNR arrays. The fabrication process is the same until the formation of the seed layer.

The electrode specimen was connected to the PCB for connection to the

measurement module. The test specimens between different concentrations of

standard solutions were washed with D.I water. From low to high concentrations of

standard solutions were measured.

Figure 6-8. Frequency sweep result for GNR modified interdigitated electrode. The unit of the standard solution is µS/cm

Figures 6-7 and 6-8 show the results of the planar electrode and the GNR-modified

112

electrode, respectively. It was confirmed that the low cut off frequency was found in

the lower region in the GNR formed electrode. As the GNR on the electrode is

generated, the surface area is significantly wider than that of the planar electrode,

resulting in an increase in EDLC . Significant improvement can be anticipated if the

entire electrode surface was coated with GNR as compared to the partial coating

obtained on the current electrode. Figure 6-9 shows the experimental results of 0.5

mS cm standard solution. It can be seen that the frequency characteristic of the GNR

electrode having a surface area wider than that of the planar electrode is formed in a

lower region.

Figure 6-9. Frequency sweep result for GNR modified electrode vs. Planar electrode. The unit of the standard solution is µS/cm

113

6.3 Conclusion

I developed a GNR integrated electrode cell based on MEMS technology. Nano

technology using AAO has been developed by many researchers [5-10 ]. Compared

with other technologies, it is easy to apply to semiconductor technology and has the

advantage of maximizing the surface area effectively. The fabrication process of

nanostructures has been combined with MEMS technology and confirmed its

possibility. Although not formed on the entire surface of the electrode, it can be

technically overcome. The surface area was maximized by forming the nanostructure

on the electrode according to the target approach, and the frequency decreasing

tendency was confirmed by testing.

114

6.4 References

[1] Wei, A., Sun, X. W., Wang, J. X., Lei, Y., Cai, X. P., Li, C. M., ... & Huang, W.

(2006). Enzymatic glucose biosensor based on ZnO nanorod array grown by

hydrothermal decomposition. Applied Physics Letters, 89(12), 123902.

[2] Zhang, W., Ganesh, N., Block, I. D., & Cunningham, B. T. (2008). High

sensitivity photonic crystal biosensor incorporating nanorod structures for enhanced

surface area. Sensors and Actuators B: Chemical, 131(1), 279-284.

[3] Fan, J. G., & Zhao, Y. P. (2008). Gold-coated nanorod arrays as highly sensitive

substrates for surface-enhanced Raman spectroscopy. Langmuir, 24(24), 14172-

14175.

[4] Zhao, Y., Zhao, Y., Hu, S., Lv, J., Ying, Y., Gervinskas, G., & Si, G. (2017).

Artificial Structural Color Pixels: A Review. Materials, 10(8), 944.

[5] Wen, L., Xu, R., Mi, Y., & Lei, Y. (2017). Multiple nanostructures based on

anodized aluminium oxide templates. Nature nanotechnology, 12(3), 244-250.

[6] Chu, C. W., Huang, Y. C., Tsai, C. C., & Chen, J. T. (2015). Wetting in nanopores

of cylindrical anodic aluminum oxide templates: Production of gradient polymer

nanorod arrays on large-area curved surfaces. European Polymer Journal, 63, 141-

148.

[7] Guo, D. L., Fan, L. X., Wang, F. H., Huang, S. Y., & Zou, X. W. (2008). Porous

anodic aluminum oxide Bragg stacks as chemical sensors. The Journal of Physical

Chemistry C, 112(46), 17952-17956.

115

[8] Rahman, S., & Yang, H. (2003). Nanopillar arrays of glassy carbon by anodic

aluminum oxide nanoporous templates. Nano Letters, 3(4), 439-442.

[9] Hwang, S. K., Lee, J., Jeong, S. H., Lee, P. S., & Lee, K. H. (2005). Fabrication

of carbon nanotube emitters in an anodic aluminium oxide nanotemplate on a Si

wafer by multi-step anodization. Nanotechnology, 16(6), 850.

[10] Masuda, H., & Satoh, M. (1996). Fabrication of gold nanodot array using anodic

porous alumina as an evaporation mask. Japanese Journal of Applied Physics,

35(1B), L126.

116

Chapter 7. Conclusion

I have developed a minimally invasive microscale needle sensor based on

semiconductor processing technology. The insertable microneedle is intended to

measure the real-time variation of electrical conductivity in cucumber stem. In

agriculture, electrical conductivity is used to measure the concentration of nutrients

supplied in plant cultivation. The concentration of nutrients, or salinity, is an

important factor in controlling the yield and quality of crop. Excess salinity raises

the concentration of the soil or hydroponic which limit the water absorbed by the

plant via osmosis. As a result, crop yield is reduced. So far, indirect electrical

conductivity (EC) measurements have been made outside the plant. Plant internal

information is needed for more accurate understanding of the plant’s physiological

conditions and to optimize the plant management system.

Internal EC information should be measured by directly accessing xylem and

phloem in the plant stem. In order to observe the microscopic structures, a novel

design has been proposed for applying electrical conductivity measurement system

to plant stem. To compensate for the limited sensor area on the narrow needle, the

interdigitated electrode design with short current and high density was used. The

width of the inserted needle is limited to 400 µm. In order to construct an array sensor

capable of multiple measurement, the space utilization was optimized by using the

two-electrode measurement method. The measurable range of conducting solution

was optimized using the cell constant. Cell constant was determined by two methods.

First approach was based on capacitance evaluation, and second approach utilized

commercial simulation tool (COMSOL).

117

Second, a micro-sized needle was fabricated to achieve the initial goal of

minimally invasive structure. The finished needle was inserted in the plant stem

without significant resistance, and the invasion level was very low compared to other

devices. A process was developed to integrate the microneedles with the designed

electrodes and a frequency evaluation was carried out to confirm the measurement

performance. The change in impedance value according to the concentration gradient

was observed. The interference between the electrode and plant tissue was modeled

and simulated. This interference phenomenon was solved by introducing the SU-8

structure. The thickness of this structure was determined by calculating the electrical

interference range.

Third, packaging technology was developed for miniaturization of the device.

The AD5933 impedance analysis chip is a total solution chip incorporating a

temperature sensor and a frequency generator. A module capable of multiple

measurements around the chip was designed and packaging technology was

developed to facilitate insertion into plants. The measured impedance was digitized

and sent to a microcontroller and connected to a computer via wireless

communication to calculate the electrical conductivity value. Sensor response to

real-time concentration changes was immediate, and noise due to diffusion was

found, but it is not a problem in plant conduits where flow is present.

Fourth, the complete sensor module was developed for measuring plant

physiological data. The target plants were selected as cucumber in consideration of

accessibility and rapid growth rate. In order to confirm the sensor error due to

temperature, the internal temperature of the plant was analyzed and it was confirmed

that the internal temperature change was negligibly small compared to the outside.

118

Two important experimental results were derived from real-time monitoring

experiments. The microneedle sensor allows selective measurement of a specific site.

Based on the results of the measurement, periodic changes in xylem and phloem

were observed. In addition, changes in ion concentration in the plant were observed

according to the concentration of external supply solution.

Fifth, nanostructures were integrated to improve sensor performance. Achieving

a micro sensor has enabled many advantages, including minimal invasion and

selective measurements. However, the effect of reducing the electrode area due to

the miniaturization of the sensor results in a negative effect. An error due to the

polarization phenomenon and a reduction in the measurement range may occur. A

GNR integrated electrode has been proposed to maximize the electrode. An

integrated manufacturing process has been developed and the performance increase

was confirmed through sample production.

The existing technology allows me to measure the ionic concentration of plant

stem using chemical analysis. But such technology is destructive and plants must be

sacrificed. Micro needle sensors manufactured with the developed electrical

measurement system successfully measured the cyclic characteristics of the ions in

the plant through real-time simultaneous monitoring. In hydroponic cultivation, the

nutrient solution discharged from the slab should be analyzed and the yield and

quality of the plant fruit evaluated to optimize the plant cultivation environment.

Currently available techniques are complex and less effective in managing the

amount of fertilizer waste in hydroponics systems. Internal measurements can

determine the level of nutrient solution that is accepted through the roots of plants

and can be used to effectively control fertilizer. My technology can contribute greatly

119

to the understanding of plant physiology and to generating higher yields and higher

quality crops.

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Appendix

A. Fabrication method for nanostructures

A.1 Anodized aluminum oxide

Anodized aluminum oxide (AAO) is a porous structure in which nano-sized holes

are formed perpendicular to the surface. AAO is used as a template for

manufacturing various molecular filters [1-2] or nanorod structures [3-4]. In this

study, a wafer-scale AAO processing system was developed and applied.

Figure A-1. Process setup for making aluminum oxide. The PEEK zig has chemical resistance and is designed to be suitable for processes using strong acids. A voltage is applied between the electrode inserted inside the zig and the electrode leading to the electrolyte, and oxidation phenomenon occurs.

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The system setup is shown in Figure A-1. I fabricated a zig with Polyether ether

ketone (PEEK) for the wafer processing. The detailed structure and functional

description of the PEEK zig is described in Chapter 6.2.1. Oxalic acid with 0.3 M

concentration was used as the electrolyte for oxidation process. On a custom-built

LabVIEW module, the voltage was set to 40 V and current limit to 5 A. The

processing time depends on the thickness of the aluminum film to be oxidized. In

this experiment, a thin film of aluminum with thickness of 2 μm was prepared via

vapor deposition. After the oxidation process, dilute phosphoric acid solution was

used to widen the pore size and remove residues. The phosphoric acid process time

was set to 45 minutes. After the completion of the all processes, the wafer was

cleaned with D.I. water.

Figure A-2. (a) process flow for AAO and gold nanorod array (GNR). (b) AAO filled by gold reduction reaction. (c) completed GNR after AAO template removal process

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A.2 Gold nanorod

For the fabrication of the gold nanorod structure, thin film of titanium, gold,

titanium, and aluminum was deposited on a silicon wafer in sequence. The first

titanium layer was deposited to improve adhesion of gold to the silicon surface, and

the third layer was deposited to improve adhesion between gold and aluminum. The

gold layer acts as the seed layer in the reduction process. Gold cyanide was the

electrolytic solution of choice for the reduction process.

The GNR structure fabrication process involves the AAO fabrication as shown in

Figure A-2 (a). After the AAO is formed, gold nanorod is grown from the seed layer

through reduction process. The shape of the gold structure is determined by the

template AAO. Figure A-2 (b) shows the reduction of gold in the AAO. When the

AAO structure is dissolved using a diluted phosphoric acid solution, a bar-shaped

array with nanoscale diameter can be obtained as shown in Figure A-2 (c).

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B. LabVIEW script for anodization process

The AAO or gold nanorod fabrication system by oxidation or reduction was

controlled by a LabVIEW program. The computer and system power supply N5771A

were connected via GPIB (general purpose interface bust) cable for communication,

and the positive and negative terminals were connected to the custom-made 4" wafer

zig with wires.

Figure A-3 shows the operation panel of the anodizing and electroplating system.

The control panel displays graphs for plotting current vs. time. To operate, first

connect the port and select the voltage level optimized for this experiment. The

current limit was set to 5 A, and over voltage protection (OVP) limit and the

sampling time were set to 10 and 1, respectively. Then, activate the knob on the

control panel to the enable state and press the program execution key to beginning

the process. The block diagram of system is shown in Figure A-4.

Figure A-2. Front panel for current measurement system (anodizing ver.)

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Figure A-4. Block diagram for anodizing or electroplating system

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C. LabVIEW script for frequency sweep measurement

Frequency sweeping system was operated based on impedance measurement with

LCR meter. The LCR meter and computer were connected via GPIB cable.

Figure A-5 shows the front panel of the impedance measurement system. The

control panel displays two graphs to plot the impedance and phase angle over time.

To operate, 1) connect the port and select the target R-X for the experiment, 2) enter

the rate of increase for sequential frequency increments, and 3) input the maximum

frequency, sampling rate, and voltage information.

Figure A-5. Front panel for impedance measurement system (frequency sweep ver.)

The block diagram of the impedance measurement system is shown in Figure A-

6. I used sequence and while loop to gradually change the operational frequency.

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Figure A-6. Block diagram for impedance measurement system with sequential incensement of frequency

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D. Appendix reference

[1] Yang, S. Y., Ryu, I., Kim, H. Y., Kim, J. K., Jang, S. K., & Russell, T. P. (2006).

Nanoporous membranes with ultrahigh selectivity and flux for the filtration of

viruses. Advanced materials, 18(6), 709-712.

[2] Osmanbeyoglu, H. U., Hur, T. B., & Kim, H. K. (2009). Thin alumina nanoporous

membranes for similar size biomolecule separation. Journal of Membrane Science,

343(1-2), 1-6.

[3] Huang, Z., Meng, G., Huang, Q., Chen, B., Zhu, C., & Zhang, Z. (2013). Large-

area Ag nanorod array substrates for SERS: AAO template-assisted fabrication,

functionalization, and application in detection PCBs. Journal of Raman

Spectroscopy, 44(2), 240-246.

[4] Zhou, Z. F., Zhou, Y. C., Pan, Y., & Wang, X. G. (2008). Growth of the nickel

nanorod arrays fabricated using electrochemical deposition on anodized Al templates.

Materials Letters, 62(19), 3419-3421.

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Abstract in Korean (국문초록)

마이크로바늘 센서 이용 식물체 주요 생체정보(전기전도도)

측정 기술 개발

식물 재배에서 전기 전도도 측정 시스템은 양분 용액과 토양의 염분을

관리하는데 사용되어왔다. 최근 연구에 따르면 원예 작물 (예: 오이, 토

마토)의 품질과 생산량은 양분 용액의 염분을 조절하여 최적화 할 수 있

다. 하지만 품종, 양분 용액의 전기 전도도, 온실 환경 그리고 재배 기술

간의 상호 관계는 매우 복잡하며 깊게 이해되지 못했다. 재배 환경에 대

한 식물의 상세한 반응 정보를 확인하기 위해서는 과육이 완전히 자라거

나 식물 자체를 희생하여 분석하는 방법을 사용해야 한다. 이 문제를 극

복하기 위해서는 실시간 모니터링을 통한 식물 생체 내부 정보(EC) 측

정 장치가 필요하다.

본 연구에서는 식물체 내부 정보를 측정하기 위한 마이크로바늘 프로

브 시스템을 제안한다. 물관과 체관은 식물의 양분 및 수분을 운송하는

기관이다. 마이크로 바늘 센서는 물관과 체관까지 삽입하여 흐르는 수액

의 전기 전도도를 측정한다. 식물이 뿌리로부터 흡수하는 수분 및 미네

랄의 농도 그리고 내부 생리 작용에 의한 이온 변화를 측정하고자 한다.

식물체 내부 생체 정보와 기존 외부 측정 정보를 통합한다면 효율적인

염도 관리를 통해 식물 생육 환경의 최적화가 가능할 것이다.

129

마이크로바늘 센서의 제조 공정은 마이크로 스케일 증착, 사진식각 및

딥 에칭 공정과 같은 실리콘 기반 기술을 포함한다. 또한, 마이크로 스

케일 제조는 모든 기능 요소가 면적 허용 범위를 충족시키고 최소한의

식물 침입으로 매우 정확하게 측정할 수 있게 한다. 식물의 물관과 체관

은 단일 크기가 마이크로 스케일이며 관다발 형태를 가진다. 따라서 내

부 측정 공간이 제한되며 fringe 효과에 따라 측정 오차가 발생할 수 있

다. Interdigitated 전극은 전극 사이에 짧은 전류 분포을 가지며 전극 면

적의 효율적인 배치로 인해 매우 작은 센서 플랫폼에 적합하다. 전극간

의 거리 및 폭은 셀 상수를 계산하여 설계하였으며, 임피던스 측정 장치

를 이용하여 평가되었다.

식물에 삽입 할 수 있는 시스템을 개발하기 위해서는 몇 가지 중요 요

소를 고려해야한다. 첫째, 식물 줄기 내부 수액의 전기 전도도 측정에

영향을 미치지 않도록 전극을 식물의 내부 조직과 분리해야한다. 금속

전극을 사용한 전기 전도도 측정 시, 전극 표면에 형성된 전기 이중층과

fringe 효과에 의해 식물 내부 조직과의 접촉할 경우 측정 된 전기 전도

도 값이 저평가된다. 본 연구에서는 전극을 보호 할 수 있는 절연층 구

조를 제안하고 시뮬레이션을 통해 설계하고 실험을 통해 검증 하였다.

둘째, 전극 표면적 극대화를 통한 센서 성능의 최적화 과정이 요구된

다. 측정 전극의 면적은 측정 범위 극대화 및 분극 현상 감소와 밀접한

관계를 가지고 있다. 본 연구에서는 전극 표면에 금 나노막대(GNR)을

형성함으로써 측정 범위의 증가 및 분극 현상의 감소를 통한 센서 최적

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화를 제안하였다. 전극 표면적 당 4um GNR을 형성할 경우 표면적 약

100배 증가 효과가 있다.

개발 된 마이크로바늘은 임피던스 분석 칩을 기반으로 제작된 측정 시

스템과 통합되었다. 측정 모듈에서 변환된 디지털 신호는 마이크로컨드

롤러를 통해 컴퓨터와 무선으로 연결된다. 통합 시스템을 이용하여 온실

에서 재배된 오이의 실시간 전기 전도도를 모니터링 하고 가능성을 확인

하였다. 이 장치는 원예 용품 (파프리카, 토마토 등)에 적용 할 수 있도

록 고안되었다. 그러나 본 연구에서는 오이를 이용한 장치 및 기본 데이

터 분석에 초점을 두었다.

키워드: 전기 전도도, 미세 바늘, 실시간 모니터링, 시설원예, 최소침습,

식물재배

학생 번호: 2009-23796