生物官能基化奈米鑽石性質及其應用

1

國 立 交 通 大 學

應用化學系博士班

博 士 論 文

生物官能基化奈米鑽石性質及其應用

Properties of Bio-functionalized Nanodiamond and Its

Applications

研 究 生：劉昱麟

指導教授：孫建文 博士

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生物官能基化奈米鑽石性質及其應用

Properties of Bio-functionalized Nanodiamond and Its

Applications

研 究 生：劉昱麟 Student：Yu-Lin Liu

指導教授：孫建文 博士 Advisor：Dr. Kien-Wen Sun

國 立 交 通 大 學

應用化學系博士班

博 士 論 文

A Thesis Submitted to Ph.D program, Department of Applied Chemistry

College of Science National Chiao Tung University

in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy

in

Applied Chemistry

September 2011

Hsinchu, Taiwan, Republic of China

I

Properties of Bio-functionalized Nanodiamond and

Its Applications

Student: Yu-Lin Liu Advisor:Dr. Kien-wen Sun

Ph.D program, Department of Applied Chemistry

National Chiao Tung University

Abstract

Diamond with good photostability, easy surface-modification, and low cytotoxicity

is a good nanomaterial for bio-applications. In the first part of this thesis, we develop

a new technique which is combination of SAM and E-beam lithography to pattern

nanodiamond on silicon base substrate. The technique which we demonstrated here is

suitable for applications of bio-sensing chips and single bio-molecule patterning and

detection. For the further works, we expect to develop for bio-chip base on

bio-functionalozed nanodimond arrays.

In the second part of this thesis, we couple nanodiamond with gold nanoparticles of

different sizes by using two complementary DNA sequences. After the gold

nanoparticles were hybridized on the nanodiamonds, we observed enhancement of the photoluminescence (PL) signals originated from nanodiamond’s nitrogen-vacancy

II

center. The enhancement was attributed to the plasmon field created by the gold

nanoparticles. The shape of the enhanced PL spectra was also affected by the sizes of

the attached nanoparticles due to their different resonant plasma frequency. The signal

III

致謝

在交通大學五年多的博士生涯即將告一個段落，在這五年多的日子

之中，一轉眼間就到了結束的時候，不管是生活上以及學業上，都受

到許多人的幫助以及照顧，在此感謝所有對我有幫助的人。

首先，我要感謝我的指導教授孫建文教授，在研究上老師總是給我

們很大的自由及發揮度，去激發出獨立思考以及解決問題的能力，並

提供我們沒有後顧之憂的研究環境，並且在實驗上適時的給予指導，

在最後不管是期刊還是論文給予適當的建議。

另外，我要感謝林諭男教授、張立教授、孫允武教授、黃振昌教授、

廖奕翰教授，在百忙之中抽空擔任我的口試委員。

接著我要感謝奈米科技的工作人員們，感謝李良箴博士在不吝嗇的

傳授實驗上的技術，使得我的實驗可以更輕易的完成。劉翊筑小姐，

在許多的公務以及生活事務上給予大力的幫忙，另外還有徐武達先生、

鍾宜娟小姐、賴良一先生...等，給予實驗上以及生活事務的幫助。

還有要感謝孫建文實驗室的伙伴們，克瑜、鏡堯、承翰，雖然跟你

們自一起做實驗的時間不常，但是從一開始的盡心盡力儀器的教學，

以及實驗室事務上的幫助。昭凱，感謝你在實驗上大力的幫忙、從一

開的的 PECVD 以及後來的 FIB...等的製程上都給予很大的幫忙，還

IV

有在資格考上指點一條明路。以及老柯，在使用 Confocal、E-GUN 以

及 PECVD...等儀器上以及在理論的討論上給予很大的幫助。感謝昭

凱與老柯在我博班生涯中一起相互努力成長。

還有實驗室其他的學弟妹，柏帆、宇涵及詩翔，感謝你們陪我一起

掉進奈米鑽石這一條不歸路。政元除了在實驗上的幫忙之外。在球場

上一起揮殺灑的日。子漢、建宏(KB)、忠儀、庭源(龍五)、晟嘉、琇

雅、靜宜、文駿、志昇、新欽、夢勳、學亨、允中...等其他學弟妹、

在這五年的博士生活感謝與我一同成長

還有感謝在實驗上幫忙的實驗室:交大應化李耀坤實驗室、交大電

子李建平實驗室、中原物理沈志霖實驗室、交大生物張家靖實驗...

等。

還有感謝我的女朋友俊儀，感謝妳一路以來的包容與陪伴。

最後感謝我的父母，感謝他們一路的支持與關心，你們是支持我的

一大力量。

有太多太多的感謝是無法用筆墨寫出的，這一刻我只能用最簡單的

文字，來回謝這些默默在我背后支持的你們，感謝你們。

V

Content

Abstract ... I 致謝 ... III Content ... V List of Figure ... VIII

Chapter 1 Introduction and Background ... 1

1-1 Diamond ... 1

1-2 Synthesize of diamond ... 2

1-2-1 HTHP (high temperature and high pressure) diamond ... 2

1-2-2 CVD (chemical vapor deposition) diamond ... 3

1-2-3 Detonation of explosive ... 4

1-2-4 Ultrasound cavitation ... 5

1-3 Impurities in diamond ... 5

1-3-1 Classification of diamond ... 6

1-3-2 Analysis of defects in diamond ... 7

1-3-3 Color center of diamond ... 9

1-4 Application of diamond ... 12

1-4-1 Nanodiamond as bio-marks ... 12

1-4-2 CVD diamond for bio-chip applications ... 14

1-4-3 Diamond for Single-photon source ... 15

1-5 Research motivation and object ... 16

1-6 Reference ... 17

Chapter 2 Skeleton of this thesis and instrumentation ... 27

2-1 Description of the remaining chapters ... 27

2-1-1 Protein Functionalized Nanodiamond Arrays ... 27

2-1-2 Plasmon-enhanced photoluminescence from bioconjugated gold nanoparticle and nanodiamond assembly ... 28

VI 2-2 Instrumentation ... 28 2-2-1 E-beam lithography ... 28 2-2-2 Confocal microscopes ... 29 2-2-3 Raman spectrum ... 30 2-2-4 Luminescence ... 32 2-3 Reference ... 34

Chapter 3 Application of bio-functionalized nanodiamond arrays... 40

3-1 Literature review ... 40

3-1-1 Nanodiamond ... 40

3-1-2 Bio-chip ... 41

3-2 Fabrication of bio-functionalized mamodiamond arrays by self assemble monolayer . 43 3-2-1 Preparation of carboxylated nanodaimond solution ... 43

3-2-2 Fabrication of nanodiamond nanoarray by self assemble monolayer ... 44

3-2-3 Fabrication of PLL and FITC functionalized nanodiamond nanoarray ... 46

3-2-4 Fabrication of Lysozyme functionalized nanodiamond arrays ... 46

3-3 Results and discussion ... 47

3-3-1 Carboxylated nanodiamond ... 47

3-3-2 Nanodimond nanoarray ... 48

3-3-3 PLL and FITC functionalized nanodiamond arrays. ... 50

3-3-4 Lysozyme functionalized nanodiamond arrays ... 51

3-4 Summary ... 53

3-5 Reference ... 54

Chapter 4 Photoluminescence enhancement of bioconjugated gold nanoparticle and nanodiamond assembly ... 71

4-1 Literature review ... 71

4-2 Fabrication of bioconjugated gold nanoparticle and nanodiamond assembly... 72

VII

4-2-2 Conjugation of gold nanoparticle with nanodiamond ... 74

4-3 Results and discussion ... 75

4-3-1 Gold nanoparticle and nanodiamond assembly ... 75

4-3-2 Optical properties ... 77

4-3-3 Photoluminescence enhancement of Au-DNA-ND assembly ... 78

4-4 Summary ... 82

4-5 Reference ... 82

Chapter 5 Conclusion ... 96

Publications ... 97

Appendix-Fluorescent defect centers in nitrogen-doped ultrananocrystalline diamond films generated by microwave plasma ... 98

A-1 Literature review ... 98

A-1-1 Ultrananocrystalline diamond films ... 98

A-1-2 Semiconductor diamond ... 99

A-1-3 Ion implantation ... 100

A-1-4 Silicon vacancies ... 102

A-2 Growth of nitrogen-doped UNCD films ... 103

A-3 Properties of nitrogen-doped UNCD films ... 104

A-4 Properties of nitrogen-doped UNCD films treated with microwave plasma ... 106

A-5 Summary ... 112

VIII

List of Figure

Figure 1-1(a): Raman spectra of three different CVD diamond films ... 24

Figure 1-1(b): The different spectra of various wavelength of laser excitation of diamond ... 24

Figure 1-2: Raman spectra of nanodiamonds of different size ... 25

Figure 1-3 (a): Photoluminescence spectra of different sizes nanodiamonds obtained by using 488nm laser excitation ... 25

Figure 1-3(b): Photoluminescence spectra of different sizes nanodiamonds obtained by using 532nm laser excitation ... 26

Figure 2-1: Overlay of electron beam system and the electron optical column of electron beam system... 35

Figure 2-2: Illustration and simulation result of forward and back scattering effect and the charge effect during the electron beam process ... 36

Figure 2-3: Illumination of confocal microscope ... 37

Figure 2-4: Energy diagram for Rayleigh, Stokes, and anti-Stokes scattering ... 38

Figure 2-5: Simplified Jablonski diagram with absorbance, internal conversion, fluorescence, intersystem crossing, and phosphorescence ... 39

Figure 3-1: Cytotoxicity test of nanodiamonds ... 57

Figure 3-2: Photostability test of nanodiamond ... 57

Figure 3-3: Synthetic route for functionalized nanodiamonds ... 58

Figure 3-4: Electric properties of surface modified silicon nanowire were changed under the variation of pH value ... 58

Figure 3-5 (a): Schematics of the functionalized NDs bonded to the SAM substrates ... 59

Figure 3-5 (b): Schematics of flow chart of the template fabrication processes ... 59

Figure 3-6: Schematic proceeding of the FITC functionalized NDs ... 60

Figure 3-7 (a): Raman spectra of NDs with and without the acid treatment61 Figure 3-7 (b): Raman spectra of NDs with and without the acid treatment ... 61

Figure 3-8: FTIR spectra of NDs with and without the acid treatment ... 62

Figure 3-9 (a): SEM images of one of the corners of the cross marks ... 63

Figure 3-9(b): SEM images of the nano holes array ... 63 Figure 3-10(a): SEM images of the cross-section of a patterned single

IX

nanodiamond ... 64

Figure 3-10(b): AFM images of patterned single nanodiamond arrays ... 64

Figure 3-11(a): Raman spectra of pattern-in and pattern-out area ... 65

Figure 3-11(b): PL spectra of pattern-in and pattern-out area ... 65

Figure 3-12(a): Optical image of the nanodiamon arrays of 1D mapping area, indicated by the straight line ... 66

Figure 3-12(b): Image of the 1D Raman intensity mapping ... 66

Figure 3-13(a): Optical image of nanodiamond arrays of the 2D mapping area, indicated by the square ... 67

Figure 3-13(b): Image of the 2D Raman intensity mapping ... 67

Figure 3-14(a): Optical image of the 1D mapping area, indicated by the straight line... 68

Figure 3-14(b): Image of the 2D PL intensity mapping. ... 68

Figure 3-15: IR spectra of three different samples (a) cND (b) lysozyme and (c) cND-lysozyme chip ... 69

Figure 3-16: Raman spectra of three different samples (a) lysozyme (b) lysozyme-cND complex in the solution (c) cND-lysozyme chip ... 69

Figure 3-17(a): SEM images of E.coli interaction with ND–lysozyme film on crossmark ... 70

Figure 3-17(b): SEM images of E.coli interaction with ND–lysozyme film on nanoarrays ... 70

Figure 4-1: ICG dye molecules were enhanced by metallic nanoparticles .. 86

Figure 4-2: Spectra of Healthview Nucleic Acid Stain, and ND-DNA1 and Au-DNA2 solutions were allowed to react with Healthview Nucleic Acid Stain... 86

Figure 4-3: Scheme of conjugation gold nanoparticle and nanodiamond by DNA. ... 87

Figure 4-4(a): SEM images of the hybrid structures of NDs coupled with the 13 nm gold nanoparticles, ... 87

Figure 4-4(b): SEM images of the hybrid structures of NDs coupled with the 30 nm gold nanoparticles, ... 88

Figure 4-4(c): SEM images of the hybrid structures of NDs coupled with the 50 nm gold nanoparticles, ... 88

Figure 4-4(d): SEM images of the hybrid structures of NDs coupled with the 80 nm gold nanoparticles, ... 89

Figure 4-5: SEM image of NDs interacted with gold nanoparticles without DNA ... 89 Figure 4-6: HRTEM image of a 30 nm gold nanoparticle attached on a ND

X

via a double string DNA ... 90 Figure 4-7(a): PL spectra of bare ND, gold nanoparticles, Au-DNA2

assembly, and ND-DNA1 assembly ... 90 Figure 4-7(b): Absorption spectra of 13nm, 30nm, 50nm, and 80nm Au

nanoparticles ... 91 Figure 4-8(a): Plasmon enhanced PL spectra of Au-DNA-ND hybrid

structures with gold nanoparticle sizes of 13 nm ... 91 Figure 4-8(b): Plasmon enhanced PL spectra of Au-DNA-ND hybrid

structures with gold nanoparticle sizes of 30 nm ... 92 Figure 4-8(c): Plasmon enhanced PL spectra of Au-DNA-ND hybrid

structures with gold nanoparticle sizes of 50 nm ... 92 Figure 4-8(d): Plasmon enhanced PL spectra of Au-DNA-ND hybrid

structures with gold nanoparticle sizes of 80 nm ... 93 Figure 4-9: Scheme of interaction between hybrid structures and NdeI

restriction enzyme ... 93 Figure 4-10: SEM image of hybrid structures interact with the NdeI

restriction enzyme ... 94 Figure 4-11: PL spectra of bare ND, Au-DNA-ND hybrid structures before

and after interacting with NdeI restriction enzyme ... 94 Figure 4-12: Time-resolved spectra of bare NDs, ND-DNA1 assembly, and

Au-DNA-ND hybrid structures... 95 Figure A-1: Conductivity data obtained in the temperature range 300–4.2 K

for a series of films synthesized using different nitrogen concentrations. ... 120 Figure A-3: Silicon vacancies generated by ion implantation ... 120 Figure A-3: Neutral vacancy was formed after nitrogen ion implantation.

Then annealing the neutral vacancy is converted into nitrogen vacancy. ... 121 Figure A-4: HRTEM image of the N-doped UNCD ... 122 Figure A-5: Raman spectra of UNCD and nitrogen doping UNCD ... 122 Figure A-6(a): EDS spectrum which the electron beam aims at the grain

boundaries ... 123 Figure A-6(b): EDS spectrum which the electron beam aims at the grains

... 123 Figure A-7: TEM image of the nitrogen doping UNCD after treated by

microwave plasma ... 124 Figure A-8: Raman spectra of (a) nitrogen doping UNCD and nitrogen

XI

power of 800W and total pressure of 10Torr (b) over high microwave

power and total pressure ... 124

Figure A-9(a): EDS spectrum of sample A ... 125

Figure A-9(b): EDS spectrum of sample B ... 125

Figure A-10(a): SIMS spectrum of sample A ... 126

Figure A-10(b): SIMS spectrum of sample B ... 126

Figure A-11: Photoluminescence spectra of nitrogen doping UNCD, sample A. and sample B which both treated by microwave plasma ... 127

Figure A-12 (a): SEM image of the slope generated by Focus ion beam .. 127

Figure A-12 (b): Photoluminescence intensity of Si-V traced at 738nm ... 128

Figure A-12 (c): Photoluminescence intensity of Si-V traced between 550nm and 650nm... 128

1

Chapter 1 Introduction and Background

1-1 Diamond

The diamond crystal is formed by SP3-bonding structured carbon atoms. There are

two face-centered cubic lattices in the diamond lattice shifted by a vector (a/4, a/4,

a/4). The lattice parameter a is 3.56683Å . Diamond has the greatest number density,

i.e. atoms per unit volume, of any known substance. This combined with the strength

of the carbon-carbon bond, giving rise to an extraordinarily high bond energy density.

Therefore, diamond exhibits extremely mechanical properties. Diamond is known as

the hardest natural materials scoring 10 on old Mohs scale of mineral hardness [1.1].

The high bonding energy density also leads to good chemical stability for resisting

most acid and alkalis. Unlike most electrical insulators, pure diamond shows good

thermal conductivity which can reach up to 20W/cm-K at room temperature [1.2].

The thermal conductivity of pure diamond which is roughly four times more higher

than copper is the highest of any known solid and excellent insulator with

conductivity about 1015 Ω-cm [1.3]. Diamond can also be treated as semiconductor

with a wide band gap of 5.45eV. Due to the wide band gap, diamond has high optical

transmissivity around >95% form deep UV to far IR. The wide band gap grants

2

electrons from its surface with very little applied voltage.

1-2 Synthesize of diamond

1-2-1 HTHP (high temperature and high pressure) diamond

In 1954, Hall and his co-workers in General Electric (GE) achieved their first

commercially successful synthesis of diamond under HPHT (high pressure and high

temperature) where diamond is a more stable form than graphite [1.4, 1.5]. Their

breakthrough was to use a belt press which was capable to produce the pressure over

10 GPa and temperature above 2000oC. Because of the large activation energy

required for breaking carbon-carbon bonds, graphite is not necessarily transformed

into diamond, even under high pressure and temperature [1.6, 1.7]. The molten iron,

nickel or cobalt which acted as solvent-catalyst was dissolved in graphite and

accelerated its conversion into diamond. In addition, the morphology of the grown

crystals is determined by the pressure and temperature. Furthermore, the growth rate

of diamond depends on the solubility of carbon on graphite-metal and metal-graphite

interface. Therefore the concerns for the features of the stable HPHT conditions were

kept to grow high-quality diamonds. The mass-production and high-quality diamond

3

1-2-2 CVD (chemical vapor deposition) diamond

In the 1950s, a domain of research performed in the Soviet Union and U.S. focused

on pyrolysis of hydrocarbon gases at the relatively low temperature at 800oC. This

low-pressure process is known as chemical vapor deposition (CVD). Since the early

1980s, this method has been the subject of intensive worldwide research. The

advantages of CVD diamond growth include the ability to produce diamond over

large areas and on various substrates, and its well control over chemical impurities.

The CVD process does not require pressure as high as HPHT.

To activate the carbon-containing precursor molecules in the CVD process, the gas

typically must reach a temperature exceeding 2000oC. To achieve this target, several

techniques can be employed, including hot filament, plasma-assisted (DC or

microwave), combustion flame, and so on activation. Because hot-filament CVD is a

simple growth method, it has been widely accepted among diamond researchers in the

early stages of research [1.8, 1.9].

The crucial fact for the growth rate and crystal quality is not only the gas

temperature, but also the gas composition, which typically use the mixture of CH4 and

H2. During the diamond growth which is under low pressure, the graphite phases are

formed on the growth surface in a certain amount which is in accordance with the

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the diamond CVD. For keeping the diamond phase stable, the atomic hydrogen has

gotten the function to etch the graphite phases selectively. The carbon source gas must

therefore be highly diluted with hydrogen. The hydrogen atoms which are

characteristic of diamond CVD under low pressure are believed to play crucial roles

in the process.

Depending on the types of substrate, there are two principal synthesis varieties: the

growth of diamond on diamond (homoepitaxy) and on non-diamond substrate

(heteroepiyaxy). Homoepitaxy growth results in monocrystalline layers with superior

properties. However, the growth rate is slow due to the low surface chemical activity

of diamond. Additionally, the diamond substrate is expensive and limited in size. In

the growth of heteroepiyaxy, films may consist of oriented or non-oriented grains.

They contain numerous defects, like grain boundaries and amorphous carbon, but can

be grown to large sizes.

1-2-3 Detonation of explosive

The explosive detonation is the other method of diamond synthesis. The technology

was based on the detonation transformation of carbon-containing explosives with

negative oxygen balance. The product is a mixture of different kinds of carbon,

carbon black, etc; the major product among them is ultra-dispersed diamond (UDD)

5

absorption ability and high reacting capacity [1.10]. Nevertheless, it is not able to

control the production more precisely in diamond size.

1-2-4 Ultrasound cavitation

Micron-sized diamond crystals can be synthesized from a suspension of graphite,

which is in the organic liquid at atmospheric pressure and room temperature, by using

ultrasonic cavitation. The diamond yield is about 10% of the initial graphite weight.

The estimated cost of diamond produced by this method is comparable to the HPHT

method; the crystalline perfection of the product is significantly worse to the

ultrasonic synthesis. This technique requires relatively simple equipments and

procedures, however, it has only been reported by two research groups, and has no

industrial use as of 2009 [1.11]. Numerous process parameters are not yet optimized,

such as, the preparation of the initial graphite powder, the choice of ultrasonic power,

synthesis time and the solvent. However, it does leave a route for potential

improvement of the efficiency and reduction of the cost of the ultrasonic synthesis.

1-3 Impurities in diamond

Pure diamond crystal is colorless. Colored diamonds contain crystallographic

6

coloration. Intrinsic defects include vacancies, interstitials and extended structural

defects. Small foreign elements, such as nitrogen and boron, can appear as single

atoms at lattice sites, while large impurity atoms tend to form complexes with other

impurities or vacancies. Nevertheless, more than 300 optical centers due to the

element H, He, Li, B, N, NE, S, Si, P, Ti, and so on in diamond are documented

[1.12].

1-3-1 Classification of diamond

Natural diamond can be typically classified into four types, i.e. Ia, Ib. IIa and IIb,

according to the dominant type of defect present. More than 95% of natural diamonds

belong to the type Ia. There was the highest concentration of nitrogen (up to 3000

ppm) in type Ia diamond. The aggregative nitrogen present in two major structures of

which are the A-center and B-center. The A-center consists of a pair of substitutional

nitrogen atoms in nearest neighborhood, while the B-center occurs as a complex of

four substitutional nitrogen atoms surrounding a lattice vacancy. The concentration of

nitrogen which is ranging between 150 to 600 ppm is still considerable in type Ib

diamond. Nitrogen forms dominantly in single-substitutional structure. The

single-substitutional nitrogen atoms act as donor centers in diamond. The optical

absorption of type I diamonds give rise to slightly yellow color [1.13].

7

1ppm) was defined as type II diamond. The type IIa diamond refers to the purest

crystals. Boron is one of the most dominant impurities in type IIb diamond. The only

boron related defect is single substitutional acceptor with an energy level 0.37eV

above the valence band. Therefore, the type IIb diamond shows P-type conductivity at

room temperature. Optical absorption which gives type IIb diamond the characteristic

blue color was produced by ionization of the acceptors.

The CVD diamond can contain different impurities, depending on the substrate and

mixing reaction gases. This classification is not used in CVD diamond, yet, is used for

HPHT synthesis diamond. Nitrogen is the most common contamination in the HPHT

synthesis diamond. Most diamond synthesized through the growing process by HPHT

is the type Ib. If boron was added into the growth chamber to reduce the concentration

of nitrogen, the type IIb diamond can be produced. The high temperature annealing

which lead to aggregation of single-substitutional nitrogen converse type Ib into type

Ia [1.14].

1-3-2 Analysis of defects in diamond

The Raman spectrum of diamond consists of a sharp peak at 1332cm-1, while

graphite gives a rise in the boarder peaks characterized by the D-band at 1350cm-1 and

the G-band at 1580cm-1. The width of 1332cm-1 peak line reveals that how much

8

spectra of three different CVD diamond films. The quality of diamond is clearly

distinguishable. The excitation wavelength influenced the results of Raman spectra.

The figure 1-1(b) shows the different spectra of various wavelength of laser excitation

of diamond. While IR laser was used in Raman measurement, non-diamond

components emerge in the spectra due to that the IR laser is more sensitive for

SP2-bond carbon scattering. If the Raman spectra were measured by UV laser, the

fairly good quality can be obtained [1.15].

The surface morphology and structure properties also influenced the results of the

Raman spectra. In figure 1-2, Raman spectra of nanodiamonds of different size are

presented. For the size of 5-50nm nanodiamond, characteristic D-band and G-band

were dominated. For the size of 100-500nm, the intense diamond peak at 1332cm-1

exhibited with traceable graphitic or amorphous signature [1.16]. However, for the

size larger than 50nm, the structure is usually explained as diamond polycrystal

structure with graphitic structure on surface. While the nanodiamond with size 100nm

and larger, the Raman spectra have similar characters of bulk diamond which has

strong and sharp 1332cm-1 peak. For the smaller nanodiamond, the graphitic or

amorphous structure dominated the spectra. We observed that nanodiamonds with

sizes 100 nm and larger, the Raman spectra have similar characters with sharp and

9

The same difference was observed in photoluminescence spectra.

Luminescence can be excited by either an electron beam or light. The methods are

called cathodoluminescence and photoluminescence, respectively. The Raman

scattering is often measured together with photoluminescence. Additionally, electron

beam excites different optical centers at the same time, which leads to complications

in interpretation of the spectra. By contrast, selective excitation is allowed in PL by

choosing proper excitation wavelengths. Figure 1-3 (a) and (b) depicts the

photoluminescence spectra of different sizes nanodiamonds obtained by using 488

and 532 nm excitation wavelength, respectively [1.16].

Electron spin resonance (ESR) spectroscopy is a technique for studying chemical

species that have one or more unpaired electrons. Unpaired electrons in solids have a

non-vanishing spin which is associated with a magnetic moment. Consequently, the

spin states have different energies in a magnetic field. Natural and synthetic diamond

has been extensively studied by ESR, now, this method is gaining new attention due

to the possibility of using the spin states in the defect center to implement a solid state

quantum bit at room-temperature [1.17].

1-3-3 Color center of diamond

If the excited and ground states of a defect are both located within the band gap and

10

under excitation. Those defects give the crystal a characteristic color, and are thus

called color centers. A huge variety of optical color centers which maintain more than

100 different color centers can be found in diamond owing to the wide band gap of

diamond [1.18].

A very important transition occurs between the levels M=0 and n=0, it is the

zero-phonon line. In absorption, the phonon-assisted transition takes place at energies

which is higher than ZPL; in emission, however, it is at lower energies, which leads to

a mirror symmetry relative to the ZPL. In addition, the charge states are another

important property of the color centers. Generally, different charged states give rise to

the complete spectra. In the semiconductors with shallow donors and acceptors, the

equilibrium charge state of defects is determined by the position of the Fermi-level.

Nitrogen is the most prominently known impurity, which forms the nitrogen

vacancy defect in diamond and it consists of a substitution nitrogen atom with a next

nearest neighbor vacancy. According to the impurities which are close to the

surrounding area acting as electron donor or acceptor, the defects can form two types

of states: neutral nitrogen vacancy center [(N-V)0] and negative-charged nitrogen

vacancy center [(N-V)-] [1.19-1.22].

The [(N-V)-] defect in diamond which consists of a substitution nitrogen atom with

11

annealed type-Ib diamond. The resulting N-V pair has the expected C3v symmetry. It

is established that the ground state is spin triplet and the ZPL absorption corresponds

to 3A3E transition. The excited orbital doublet state produces the characteristic

zero-phonon line at 637nm (1.945 eV) with a radiative decay time of 13ns [1.23]. The

center also produces a paramagnetic resonance signal, which are the properties

confirming the C3v symmetry.

The [(N-V)0] center has nominal C3V symmetry and zero-phonon E-A optical

transition at 575nm (2.158 eV). The remarkable point of the 575nm center is the

absorption that is to be very weak to detect, especially in the case of highly nitrogen

contained type-Ib diamond. Its lifetime is 6ns at room temperature [1.24].

It has been reported that several other color centers emitting at the wavelength of

768, 746, 749, 764, 756, and 772nm was ascribed to defects containing Ni, Si and Cr

atoms [1.25-1.29]. These centers were found in diamond nanocrystals grown by CVD

method or were made in bulk diamond by ion implantation of the corresponding

species. Even the atomic structure of these centers has not been established yet.

However, the 768nm center has been tentatively ascribed to a complex containing Ni

and Si atoms: the 746, 749, 764, 756nm centers have been ascribed to Cr-related

defects, while the 772nm center night is a defect incorporating Cr and Ni atoms. A

12

luminescence lifetime of the 749nm Cr-related center is about 1 ns, which makes it

the most efficient single-photon emitter working in bulk diamond.

1-4 Application of diamond

The research efforts in diamond synthesis are rewarded by its unique properties,

which are then useful for many applications. Diamond has various advantages for

electronics in the semiconductor. Due to the band gap, it is inherently suitable to emit

or detect UV light. The low electron affinity allows electron emission at low

temperatures. Devices based on diamond work readily at harsh conditions, e.g. under

radiation or chemical corrosion. UV-sensors[1.30], light emitting diodes[1.31], cold

cathode,[1.32] metal-semiconductor FETs,[1.33] and electro-chemical electrodes[1.34]

have all, meanwhile, been implemented using diamond.

1-4-1 Nanodiamond as bio-marks

Over the past few years, nanomaterials, a branch of nano-biotechnology with

emphasis on the views of the biological, have attracted much attention [1.35]. The

study of nanomaterials involves biological applications and fabrication of

bio-functionalized devices. Quantum dots have specifically been applied in

13

owing to the quantum yield can be compared to fluorescent dyes and have less

photobleaching [1.36]. They were widely employed for the vitro imaging of

pre-labeled cells due to those advantages. The ability to image single-cell migration in

real time is expected to be important to several research areas, such as embryogenesis,

cancer metastasis, stem-cell therapeutics, and lymphocyte immunology. However, the

bio-cytotoxicity of quantum dots was concerned on medical applications. The

semiconductor quantum dots can be dissolved, in a process as known as photolysis, to

release toxic ions into the culture medium. The quantum dots must be carefully

examined before its bio-cytotoxicity can be approved for clinical use.

In comparison, nanodiamonds which consist of carbon atoms possess remarkable

features of low bio-cytotoxicity [1.37, 1.38]. As a result, the nanodiamonds is a

relative safe nanomaterials based on its non-bio-cytotoxicity and biocompatibility.

Nanodiamonds are promising nano-materials owning to good chemical stability,

various nanosizes, biocompatibility, good surface modification and good optical

property for bio-applications [1.39-1.42]. The surface of nanodiamonds can also be a

unique platform for the conjugation of chemicals and biomolecules after functional

modifications. The surface of nanodiamond can carry a variety of oxygen containing

functional groups immediately after purification in strong oxidative acids or oxidation

14

Surface carboxylated group of nanodiamond are typical ligands used for covalent

coupling of these biomolecules to nanodiamonds through amide linkage.

The Raman[1.15] and photoluminescence[1.19-1.12] properties of

nanodiamonds have been intensively studied. Some intrinsic Raman signals can be

used as detection markers or can be employed in biological objects. This diamond

Raman peak is strong and isolated, so it can be used as an indicator for allocating

nanodiamonds.

1-4-2 CVD diamond for bio-chip applications

CVD diamond can be mono- or polycrystalline. Whereas monocrystalline diamond

requires a diamond substrate, polycrystalline diamond can be grown on different

materials, such as silicon (Si) and quartz (SiO2), and is dominated by columnar

growth. For grain sizes below 500 nm, the CVD film is called nanocrystalline

diamond (NCD); after a certain thickness, the grain size near the surface exceeds 500 nm and the film then becomes microcrystalline diamond (μ-CD). In addition, a slightly different material is ultra-nanocrystalline diamond (UNCD) which the grain

size and roughness are not dependent on the film thickness, because these films do not

show columnar growth. For optimization of detection techniques and sensitivities, for

realization of highly integrated sensor arrays and for bio-interfaces, materials like Si,

15

CVD diamond is an outstanding material for bioelectronics with good electronic and

chemical properties [1.43, 1.44]. Additionally, ultra-hard diamond (50–150 GaP) is

promising with respect to mechanical stability of nanostructure.

1-4-3 Diamond for Single-photon source

The development of reliable devices for the generation of single photons is crucial

for many applications such as, for example, quantum cryptography[1.45], optical

quantum computation[1.46] as well as experiments on the foundations of quantum

optics [1.47, 1.48]. Single-photon emission was first observed from single atoms and

ions in traps and from molecules. More recently, semiconductor quantum dots and

photoactive point defects, such as nitrogen-vacancy (N-V) and silicon-vacancy (Si-V)

in diamond have been used in SPE experiments [1.49, 1.50]. Among the array of

luminescent nanomaterials, color centers in diamond seem to be the most-promising

single-photon source for quantum-physics applications, such as optics, information

processing and cryptography. This is due to the remarkable photoemission properties

of the defects, such as their extraordinary stability at room temperature and their high

quantum efficiency (typically >0.1), which are combined with the unique chemical

and mechanical properties of the diamond material itself. Moreover, the association of

a spin structure in the ground level resulted in a long coherence time at room

16

quantum-state preparation by optical pumping and single-spin quantum state readout

[1.51].

1-5 Research motivation and object

Nanodiamond carry the following features, such as, good photostability, easy

surface-modification, and low cytotoxicity to be considered as a good nanomaterial

for bio-applications. In this thesis work, we have developed a new technique which is

a combination of SAM and E-beam lithography to pattern nanodiamond on silicon

base substrate. As a result of the good bio-molecules absorptivity of nanodiamond, the

nanodiamond arrays can easily be bio-functionalized. The technique which we

demonstrated here is suitable for applications of bio-sensing chips and single

bio-molecule patterning and detection. In what follows, we will expect to develop it to

be as bio-chip based on bio-functionalozed nanodimond arrays.

The optical property of nanomaterials plays an important role for bio-applications,

such as bio-labeling or optical sensing. The surface plasma resonance effect from the

metallic nanoparticles greatly enhances the optical property. The ability to increase

the fluorescence emission can greatly improve the effectiveness of fluorescence-based

applications, such as single molecular detection and sensing in biology. The surface

17

applied as template for bio-sensing chips. This optical enhancement can be used as

optical sensing bio-sensors.

1-6 Reference

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23

computation with linear optics" Nature, vol. 409, 46, (2001)

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demonstration of complementarity with single photons," Appl. Phys. B, vol. 76, 113,

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24

Figure 1-1(a): Raman spectra of three different CVD diamond films [1.15]

Figure 1-1(b): The different spectra of various wavelength of laser excitation of

25

Figure 1-2: Raman spectra of nanodiamonds of different size [1.16]

Figure 1-3: (a) Photoluminescence spectra of different sizes nanodiamonds obtained

26

Figure 1-3: (b) Photoluminescence spectra of different sizes nanodiamonds obtained

27

Chapter 2 Skeleton of this thesis and instrumentation

2-1 Description of the remaining chapters

This thesis is composed of two separated but closed related research works on

nanodiamonds. A short abstract of each experimental work and instrumentation

sections are given below. More details and discussion are presented in chapter 3 and 4.

Studies related to the nitrogen-doped ultrananocrystalline diamond films generated by

microwave plasma are put in the appendix attached to this thesis.

2-1-1 Protein Functionalized Nanodiamond Arrays

Nanodiamonds possess remarkable features such as low bio-cytotoxicity, good

optical property in fluorescent and Raman spectra, and good photostability for

bio-applications. In chapter 3, we devise techniques to position functionalized

nanodiamonds on self-assembled monolayer (SAMs) arrays adsorbed on silicon and

ITO substrates surface using electron beam lithography techniques. The nanodiamond

arrays were functionalized with lysozyme to target a certain bio-molecule or protein

specifically. The optical properties of the nanodiamond-protein complex arrays were

characterized by a high throughput confocal microscope. The synthesized

28

interacting with E. coli. This work is elaborated in chapter 3.

2-1-2 Plasmon-enhanced photoluminescence from bioconjugated gold

nanoparticle and nanodiamond assembly

In this part of the work, we coupled NDs with gold nanoparticles of different sizes

using two complementary DNA sequences. After hybridizing the gold nanoparticles

on the NDs, we observed the enhancement of the photoluminescence (PL) signals

originating from the nitrogen-vacancy (N-V) center of the ND. The enhancement was

attributed to the plasmon field created by the gold nanoparticles. The lineshape of the

enhanced PL spectra was also affected by the sizes of the attached nanoparticles due

to their different resonant plasma frequencies. The signal enhancement can be used as

an indexing tool for bio-sensing applications. This work is elaborated in chapter 4.

2-2 Instrumentation

2-2-1 E-beam lithography

Generally speaking , electron beam lithograph system is constructed from four

main parts ; including electron optical column、chamber、handling system、and control

29

electron. It controls the image resolution and exposing quality. Electron optical

column is generally composed of electron gun、blanking、condense lens、stigmator、

objective lens、deflector、and electron detector (figure 2-1). These accessories enable

the electron beam ejecting from electron gun to be well-controlled and expose to the

right place.

There are three common phenomena occur during the exposure of electron beam

system ; including forward scattering、back scattering、and charging effect. Each of

them can influence the quality of the e-beam exposed patterns, especially when the

pattern size is down to sub-50nm. The effect of forward and back scattering will

broaden the pitch size and produce some proximity effect. The charging effect is the

results of the electron accumulation phenomenon. These effects will blur the exposed

image and produce a poor exposure quality (figure 2-2).

2-2-2 Confocal microscopes

The principle of confocal imaging was patented in 1957 by Marvin Minsky and

aims to overcome the limitation of traditional wide filed fluorescence microscopes

[2.1]. In wide field fluorescence microscope, the entire specimen is flooded in light

from a light source. The whole specimen in the optical path was totally excited at the

same time and the fluorescence of the specimen was detected by the microscope’s

30

microscopes is to use both point-wise illumination and detection. As shown in figure

2-3, the point illumination and a pinhole were in an optically conjugate plane in front

of the detector in order to eliminate out-of-focus signals. The name confocal stems

from this configuration. As the only signals very close to the focal plane can be

detected, the image, the image's optical resolution, particularly in the sample depth

direction, is much better than that of wide-field microscopes. However, as much of

the light from sample fluorescence is blocked at the pinhole, this increased resolution

is at the cost of decreased signal intensity – so long exposures are often required.

As only one point in the sample is illuminated at a time, 2D or 3D imaging requires

scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in

the specimen. The achievable thickness of the focal plane is defined mostly by the

wavelength of the used light divided by the numerical aperture of the objective lens,

but also by the optical properties of the specimen. The thin optical sectioning possible

makes these types of microscopes particularly good at 3D imaging and surface

profiling of samples.

2-2-3 Raman spectrum

Although the inelastic scattering of light was predicted by Adolf Smekal in 1923, it

is not until 1928 that it was observed in practice. The Raman effect was named after

31

means of sunlight (1928, together with K. S. Krishnan and independently by Grigory

Landsberg and Leonid Mandelstam). Raman won the Nobel Prize in Physics in 1930

for this discovery accomplished using sunlight, a narrow band photographic filter to

create monochromatic light and a "crossed" filter to block this monochromatic light.

He found that light of changed frequency passed through the "crossed" filter.

Systematic pioneering theory of the Raman effect was developed by Czechoslovak

physicist George Placzek between 1930 and 1934. The mercury arc became the

principal light source, first with photographic detection and then with

spectrophotometric detection. At the present time, lasers are used as light sources.

The Raman effect is a light scattering phenomenon. While light of frequency V0

(usually from a laser) irradiates a sample, it can be scattered. The frequency of the

scattered light can either be at the original frequency which referred to as Rayleigh

scattering or at some shifted frequency VS = V0 Vinternal (referred at as Raman

scattering). The frequency Vinternal is an internal frequency corresponding to rotational,

vibrational, or electronic transitions. In discussing the Raman effect, some commonly

used terms need to be defined. As shown in figure 2-4, radiation scattering to the

lower frequency side (to the red) of the exciting line is call Stockes scattering. The

scattering radiation at the same frequency as the incident radiation is called Rayleigh

32

is referred to as the anti-Stokes scattering. Finally, the magnitude of this shift between

the Stokes or the anti-Stokes line and the exciting line is called the Raman shift, ΔV = ∣V0 – Vinternal ∣. The energy diagram for Stokes and anti-Stokes scattering was shown in figure 2-4.

2-2-4 Luminescence

Luminescence is the emission of light from any substance and occurs from

electronically excited states. Luminescence can be of two types: fluorescence and

phosphorescence. Phosphorescence is emission of light from triplet-excited states, in

which the electron in the excited orbital has the same spin orientation as the

ground-state electron. Transitions to the ground state are forbidden and the emission

rates are slow (103-100 s-1), so phosphorescence lifetimes are typically milliseconds to

seconds. Phosphorescence is usually not seen in fluid solutions at room temperature,

but there are many deactivation processes that compete with emission, such as

nonradiative decay and quenching processes. Fluorescence is emission light from

singlet-excited states, in which the electron in the excited orbital is paired (of opposite

sign) to the second electron in the ground-state orbital. Return to the ground state is

spin-allowed and occurs rapidly by emission of a photon. Those emission rates of

fluorescence typically are 108 s-1, so that a typical fluorescence lifetime is near 10 ns.

33

spectra vary widely and are dependent upon the chemical structure of the fluorophore

and the solvent in which it is dissolved.

A fluorophore is usually excited to some higher vibrational level of either S1 or S2.

With a few rare exceptions, molecules in condensed phases rapidly relax to the lowest

vibrational level of S1. This process, called internal conversion, is nonradiative and

takes place in 10-12 seconds or less. Return to the ground state occurs to a higher

excited vibrational ground-state level, which then quickly reaches thermal equilibrium.

An interesting consequence of emission to a higher vibrational ground state is that the

emission spectrum is typically a mirror image of the absorption spectrum of the

S0S1 transition.

Molecules in the S1 state can also undergo a spin conversion to the first triplet state,

T1. Emission from T1 is termed phosphorescence and is generally shifted to longer

wavelengths (lower energy) relative to fluorescence. Transition from the T1 to the

singlet ground state is forbidden, and as a result, the rate constants for triplet emission

are several orders of magnitude smaller than those for fluorescence. As shown in

figure 2-5, processes which occur between the absorption and emission of light are

34 2-3 Reference

[2.1] Paul Daviddovits and M. David Egger, "Scanning Laser Microscope," nature,

vol. 223, 831 (1969)

[2.2]ELS-7500EX Electron Beam Lithography System Instrument Manual, ELIONIX

Inc.

35

Figure 2-1: Overlay of electron beam system and the electron optical column of electron beam system

[2.2]

36

Figure 2-2: Illustration and simulation result of forward and back scattering effect

37

38

39

Figure 2-5: Simplified Jablonski diagram with absorbance, internal conversion,

40

Chapter 3 Application of bio-functionalized nanodiamond arrays

3-1 Literature review

3-1-1 Nanodiamond

The cytotoxicity of nano-materials has been mostly concerned while the

development of biomedical applications. Among of numerous nano-materials,

nanodiamonds possess remarkable features of low-cytotoxicity. In the previous

reports [3.1], it has been shown that nanodiamonds induce no significant cytotoxicity

in variety of cell types. Jui-I. Chao, Chia-Liang Cheng, and their coworkers has

shown that nanodiamonds are no or very low cytotoxicity for lung cells. The

cytotoxicity test of nanodiamonds in human lung cells was demonstrated (figure 3-1).

It indicates that nanodiamonds did not significantly induce the cell death. The

nanodiamonds examined in many other cells including neuronal, renal, and cervical

cells also show no cytotoxicity in those cells. As a result, nanodiamonds are relatively

safe nano-materials for further evaluation of clinical applications.

Other the low cytotoxicity, the detectable fluorescence of nanodiamonds with no

photobleaching was demonstrated in previous researches [3.2]. The figure 3-2 shows

41

(blue) excited under the same conditions. Accord to the results of photostability test,

no sign of photobleaching was found for nanodiamond even 8 hours of continuous

excitation. By contrast, the fluorescence of polystyrene nanospheres was

photobleached within 0.5 hour under the same condition.

The nanodiamonds are chemically inset, but can be surface-functionalized easily.

For the oxidative acid-treated, nanodiamonds can carry a variety of

oxygen-containing such as carboxylated group. Carboxylated nanodiamond can be

used to as a staring material to prepare other surface functionalized nanodiamond. The

figure 3-3 shows the synthetic route for functionalized nanodiamonds. It demonstrates

that the surface of nanodiamonds can be modified into kinds of functional groups [3.3,

3.4].

3-1-2 Bio-chip

The development of biochip is a major thrust of biotechnology, which encompasses

a very diverse range of research. At the same time, the semiconductor fabrication

technology has been steadily perfecting the science of micro and nano- miniaturiztion.

In recently, the combination of those two technologies has enabled biotechnology to

begin packing traditionally bulky sensing tools into smaller and smaller area named as

bio-chip. Thousands of biochemical reactions were performed at those miniaturized

42

various purposes such as disease diagnosis or detection of bio- agents.

The biochip can be typically classified into two types. One is the processing chip

and the other one is microarray chip. The processing chip, also named as Lab-on-chip

(LOC), is a device which integrates several laboratory functions on a single chip by

combination of micro-fluidics and Microelectromechanical systems (MEMs) [3.5].

For the microarray chip, the bio-molecules such as DNA, protein, or cells were

deposited on the flat substrates like glasses, silicon wafer, or polymers with the high

density and small areas. Surface chemistry is used to covalently bind the sensor

molecules to the substrate medium.

The development of biochip is multi division of research systems including

bio-molecules, chip surface modification, and signal detection. The bio-molecules

modification on surface is very important for bio-chip researches. Physical adsorption

and chemical adsorption are widely used in bio-chip. In the physical adsorption,

bio-molecules attach to substrate via Van der waals force such as hydrophobic

interaction and electrostatic attraction force. In the chemical adsorption,

bio-molecules form the covalent linking with substrate. For example, amino group of

horseradish peroxidase (HRP) reacts with carboxylated group of

16-mercaptohexadecanoic (16-MHA) forming amide bonding.

43

reference for detecting reaction of biomolecules. In the previous report, Yi Cui and his

coworkers demonstrated that the electric property of silicon nanowire was altered by

charge change of surface modified molecules. The figure 3-4 shows electric properties

of surface modified nanowire were affected by the variation of pH value [3.6].

3-2 Fabrication of bio-functionalized mamodiamond arrays by self assemble

monolayer

3-2-1 Preparation of carboxylated nanodaimond solution

The average diameter of the nanodiamond (ND) power used in our experiments is

about 100nm (General Eletric company, USA). The particle size is confirmed by SEM.

The 0.15g nanodiamond powder was treated with the 160ml 5:1 mixture of

concentrated H2SO4 and HNO3 solutions at 75 oC for 1 hour in ultrasonic bath for

dispersion and stir for 11 hours for carboxylated reaction, and extensively rinsed

several times with DI water and dry [3.7] . The sediment was then collected and dried.

The functional COOH group was formed on the ND surface followed by the standard

chemical treatment mentioned above. The surface functional carboxylated group of

nanodiamonds was checked by Fourier transform infrared and Raman spectroscopy.

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make the dissociation of water into H+ and OH- ions. While the OH- ions absorbs on

the surface of nanodiamonds, the increasing of electric charges on nanodiamond

surface induce a coulomb repulsion force between nanoparticles. Therefore, the

clustering of nanodiamonds can be avoided. The NDs solution was prepared by

adding 0.1 g of carboxylated functionalized NDs into 100 ml of deionized water

followed by an ultrasonic bath for 60 min.

3-2-2 Fabrication of nanodiamond nanoarray by self assemble monolayer

A silicon wafer was first diced into 15 mm x 15 mm chips. A silicon oxide layer

was grown on the silicon chips with a thickness of about 400 nm by using Plasma

enhanced chemical vapor deposition (PECVD). The substrate was first cleaned with

ultrasonic bath in acetone, isopropyl alcohol, and deionized water solution for 5 min.

Then the ZEP520 photoresist was spin-coated on the silicon oxide substrates at a rate

of 500 rpm for 10 sec and 5000 rpm for 50 sec, and baked at 180 oC for 2 min. The

thickness of the photoresist on the slicon chip was about 300 nm.

Self-assembled monolayer (SAM) is an organized layer of amphiphilic molecules

in which one end of the molecules, the head group shows s specific, reversible

addinity for substrates. SAMs are created by the chemical sorption of hydrophilic

head groups onto a substrate from the vapor or liquid phase followed by a slow two

45

form either a disordered mass of molecules or form a lying down phase, and over a

period of minutes to hours, begin to form crystalline or semicrystalline structures on

the substrate surface. The hydrophilic head groups assemble together on the substrate,

while the hydrophobic tail groups assemble far from the substrate. Areas of

close-packed molecules nucleate and grow until the surface of the substrate is covered

in a single monolayer. To form an amino-terminated layer on the surface of substrate,

the substrates were immersed in 5 vol% solution of 3-aminopropyl triethoxysilane

(APTES) in 95% ethanol for 4 hours and later rinsed with ethanol and thermally

treated at 120 oC for 60 min [3.10].

The patterned substrate was dipped into 3 ml of the NDs solution and 3 ml of 0.1 M

MES buffer (2-(N-morpholino) ethane sulfonic acid). After which, 6 ml of 0.025 M

EDC solution 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride, 0.025

M NHS solution (N-hydroxysuccinimide) (here after ―EDC/NHS solution‖) and 8 ml

deionized water were added into the reaction and allowed to stabilize for 8 hours.

After the reaction was completed, the substrate was washed with acetone. The entire

template was then immersed into ZDMAC (dimethylacetamide) solution for 4 hours

to remove the photoresist. The substrate was again washed with acetone and

deionized water, then dried with N2. The figure 3-5 (a) and (b) show how the