Functionalized diamond surfaces for DNA aptamer sensing of small molecules

(1)

Functionalized diamond surfaces for DNA aptamer sensing of small

molecules

February 2018

EVI Suaebah

スアエバエヴィ

(2)

Functionalized diamond surfaces for DNA aptamer sensing of small

molecules

February 2018

Waseda University

Graduate School of Advance Science and Engineering Department of Nanoscience and Nanoengineering

Research on Nanodevices EVI Suaebah

スアエバエヴィ

(3)

I

Dedication

To my parents

to my graceful daughters who was given a warmth hearth in every single breath

I love you.

(4)

II

Acknowledgement

I owe to thank to ALLAH who has made my dream comes true. Being a doctoral student is my dreams science I was in high school. I would also like to say many thanks to a number of people who have supported me throughout this incredible journey to the completion of this doctoral courses. After an intensive period of almost five years, today is the day: writing this note of thanks is the finishing touch on my dissertation. I would like to reflect of the people who have assisted and helped me so much throughout this period.

I would first like to thank my supervisor, Prof. Hiroshi Kawarada. He who was assisted me greatly and were always willing to assist me. Your excellent cooperation and all of opportunities you was given to conduct my research and all the kindness during my study in Waseda University.

In addition, I would like to thank co. supervisor Prof. Watanabe and Prof. Tanii for their valuable guidance.

I would also like to thank my parents for their wise counsel and sympathetic ear. For my family, my husband who was given a limitless support for my personal courage and give complete support when I tried finishing my study.

For my lovely daughter, who have a great smile and warm heart who always omit my jaded when I was exhausted being a student. You are always there for me. Finally, there are my friends. We were not only qualified to support each other by deliberating over our problems and findings, but also happily by talking about things other than just our papers.

Thank you very much, everyone!

Evi Suaebah

Tokyo, February 2018

(5)

III

Abstract

Currently, diamond research is continuously finding interesting ways to produce new sensing devices and, because of its unique, high-performing and excellent material, diamond is also popular in nano-sensing applications. The advantages of the diamond surface to nanotechnology has become a key role for producing high-performance sensors sensitive for small molecule detection. A brief introduction of a typical nanocrystalline diamond for a diamond biosensor will be given (Chapter 1). The challenge for producing a diamond sensing device is stability and selectivity of the surface functionalization for the biomolecular reactions that may be exposed to the diamond surface. In addition, surface functionalization is the most critical method to improve the capability of diamond to become an active region for biomolecular activity. Herein, we attempt to functionalize a diamond surface with amine and carboxyl terminations. The main purpose of functionalization research is to discover ways to produce diamond surface functionalization via the simplest and newest techniques with high performance and high stability upon biological reaction. Surface functionalization via direct amination and carboxylation is the primary method for developing an entirely diamond sensing system. Surface functionalization was conducted by different techniques for the amination and carboxylation. First, carboxylation was achieved by ultraviolet (UV) ozone cleaner and vacuum UV (VUV) treatment, while amination was achieved by the nitrogen radical beam (NRB) technique.

Diamond surface functionalization was found to be satisfactory via nitrogen/hydrogen radical beam treatment. The capability of the aminated diamond sensor was confirmed using different molecules with similar characteristics, where the molecule used was relatively small compared to a protein. Estrogen is an acceptable candidate as an aptamer target for aptasensing detection. A label-free fluorescence aptasensor for the determination of 17β-estradiol (E2) was developed based on the diamond surface functionalization via NRB for biomolecule interaction and sensing detection. The diamond

(6)

IV

surface was treated using the NRB system to produce amine termination on the diamond itself, while the sensor was fabricated by the photolithography technique to produce a dot pattern to be used as the sensing area for biomolecule activation. The immobilization of the supporting DNA and hybridization of the aptamer was designed to be used as a detection pair to bind with any E2 molecule, where the aptamer captures the E2 molecule naturally. The fluorescence microscope signal indicator was used for E2 detection and the biomolecule activity was conducted under special treatment, whereupon three different stages were detected in the fluorescence results. When the E2 was detected by the aptamer, the intensity of the fluorescence signal decreased coinciding with the binding of the aptamer with the E2 molecule. The detection of E2 was thus demonstrated by determining the fluorescence signal intensity of the dot pattern area. When the aptamer was released from the supporting DNA to form a complex with the E2 molecule, the intensity of the dot pattern decreased rapidly.

The differentiation between the hybridization and the detection signal indicated the amount of E2 molecules bound with the aptamer. The proposed method provided a simple and powerful method for small-molecule detection with high sensitivity by using the basic reaction of an aptamer. (Chapter 3.1.1)

The durability and reusability of the aminated diamond sensor is influenced by radiation and the purging technique, where the purging technique is a crucial process used to improve the amine bonding on the diamond surface. The critical purpose of the purging process is that, with it, the amine termination will activate more easily in rich gas compared to without the purging process. Without the purging process, the amine bonding was not established on the diamond surface and the immobilization was affected. Indeed, if there is no supporting DNA on the diamond surface, hybridization between the supporting DNA and aptamer become zero. (Chapter 3.1.2)

With the success of carboxyl termination as a surface modification on the diamond surface, our curiosity expanded to amine termination via a unique method using the NRB system. The novel method for micropatterning oligonucleotides on the diamond surface was reported via nitrogen/hydrogen terminating on the diamond surface and subsequent

(7)

V

hybridization. Further, the covalent bonding of the supporting oligonucleotide and the characterization of an immobilized hybridized oligonucleotide with Cy5 modification were investigated by fluorescence microscopy. The effectiveness of the two types of plasma nitrogen/hydrogen termination were investigated on diamond using both the hydrogen/nitrogen plasma treatment and the pure nitrogen plasma treatment. From the hybridization results, the hydrogen/nitrogen plasma produces amine bonding on the diamond surface, while pure nitrogen plasma produces no amine bonding on the diamond surface.

Fluorescence microscopy observed a dot pattern formation on the diamond surface via amine bonding between the diamond and biomolecules. The clearly different fluorescence imaging results confirmed that nitrogen/hydrogen radical treatment improves the amine terminals on diamond surface. In contrast, the pure nitrogen treatment results demonstrate that covalent bonding between diamond and biomolecules does not occur with this treatment (Chapter 3.2.1)

The two different treatment methods were applied on diamond to produce amine termination, where the radical treatment and the chemical treatment were used to improve and compare the capability of the diamond surface. Amine termination on the diamond surface via the NRB technique was found to be a good candidate for aminated treatment. The durability and reusability of the diamond surface for biosensing application was accomplished herein using the NRB treatment. (Chapter 3.2.2)

The capability of the diamond sensor was examined by the simplest method of biomolecule reaction on the diamond surface. This simple technique was adenosine triphosphate (ATP) detection with a direct carboxyl terminal on the diamond surface, which was confirmed by the signal-off method. The ATP detection by aptamer was conducted successfully without a labeling process, and the biomolecule was immobilized onto the nanocrystalline diamond (NCD) without any additional linker. From this result, the covalent bonding stability of the diamond and the immobilized DNA was confirmed by the washing treatment. A carboxyl coverage of about ~1% was acceptable for the immobilization process.

Indeed, the diamond together with the biomolecule is a highly functional collaboration for a

(8)

VI

biosensor, even when possessing a low coverage for carboxyl termination. The diamond sensor stability was confirmed by the reusability and reproducibility technique (Chapter 3.2.3)

The improvement of the diamond surface functionalization via carboxyl termination was conducted in high-performance mode and determined with electrical measurement. This work proposes using the simplest diamond functionalization by carboxyl termination for ATP detection by an aptamer. The high-sensitivity sensor with the label-free aptamer for ATP sensing detection was fabricated on the NCD. The carboxyl termination was generated by a VUV excimer laser, and the fluorine termination used as a passivation layer on the background area was generated by inductively coupled plasma. The ATP detection was observed as a fluorescence signal intensity decrease for 66% of the hybridization intensity signal. The sensor operation was also investigated by the field-effect characteristics, where the indicator of ATP detection was an observed shift of the drain current–drain voltage characteristics, herein measured as a 78 mV shift in the negative. The capability of the aptamer for ATP detection is thus demonstrated by the negative charge direction, and the combination of fluorescence and field-effect transistor on the modified diamond surface indicates its ability to successfully detect ATP. (Chapter 3.2.4)

(9)

VII

Chapter 1 Introduction

(12)

2

1.1 Research Background 1.1.1 Diamond Biosensor

Diamond materials exhibit high feasibility for various kinds of surface functionalization, such as hydrogen, amines, carboxyl acid, and fluorine [1-4]. This facile surface functionalization can be achieved readily to effectively control the diamond surface by chemical and physical treatment [5-8]. There are several different types of diamond materials including single-crystal diamond, polycrystalline diamond, and nanocrystalline diamond ( NCD) [9-11]. In past research, diamond has been shown to be a material with high physical stability, a wide potential window, chemical inertness, and biocompatibility; which has led to researchers positing is usefulness in the production of biosensors [12-15]. Diamond is a promising material for merging solid and biological systems [16-18], and the use of diamond as a biosensor platform fits the criteria of low price, possibility of surface chemistry modification, and potential for DNA detection [19-20]. Recently diamond has attracted the attention of many research groups because of its impressive combination of chemical, physical and mechanical properties suitable for several applications. For example, diamond exhibits a stable allotropic configuration of carbon at high temperatures and pressures, flexibility in chemical bonding, and is harmless and thus a suitable building block for applications as a bio-interface [13]. The important attribute derived from several advantages is that diamond is compatible for bio-application with high performance. This is owing to the consistency of the sp3 dangling bonding as a basis point for surface functionalization, with which the diamond can be activated as a biosensor using many various treatments [21]. The diamond surface functionalization can improve the surface electricity, conductivity and charge, and the hydrophobicity and hydrophilicity can be controlled as well [22-24].

The diamond surface functionalization is thus an important starting point to produce a sensing device for further chemical and biomolecule activities. In Figures 1.1 and 1.2, the hydrogen termination of the diamond surface was activated with a biomolecule (i.e., DNA) coupled with a TFA amine linker.

(13)

3

Fig. 1.1 (a) Diamond terminated surface by hydrogen termination and the TFA amine. (b) TFA-amine attachment by photochemical. (c) TFA-amine deprotection attachment with tetramethylammonium hydroxide ((CH3)4NOH). (d) Carboxyl group attachment. (e) The biomolecules and crosslinker coupling [18].

(14)

4

Fig. 1.2 (a) Electrochemical and (b) photochemical bonding mechanisms. (a, i–v) H- or O-terminated diamond are electrochemically bonded to Nitrophenyl linker molecules.

heterobifunctional cross-linker reacted with an aminophenyl after Nitrophenyl is reduced.

Finally, thiol-modified ss-DNA is attached. (b, i–v) H-terminated diamond covalently attached to Amine molecules after photochemically process. The heterobifunctional crosslinker and thiol-modified ss-DNA [15].

As the development of the sensor has progressed, the linker molecule is no longer needed for biomolecule activation on a diamond surface [3]. Instead, surface functionalization technology has improved to enable us to produce a direct linker on the diamond surface. Further, the carboxyl termination or amine termination is directly modified on the diamond surface. For further chemical attachment, carboxyl and amine terminations

(15)

5

are particularly useful as functional groups, whereon DNA or any kind of biomolecule can be directly attached onto the diamond surface functionalization. This development of diamond utilities has been established to produce integrated sensing and signal processing for electrical and chemical properties [18]. The major challenge for the diamond sensor is the selectivity and stability of the surface modification when interacting with biological activities.

Currently, the sensitivity and stability are not an issue for producing a good sensing device because diamond provides extremely good stability and sensitivity and is fully compatible for nanoelectronics and chemical methods [23, 25]. Figure 1.3 illustrates the sensitivity of the diamond surface functionalization for pH detection.

Fig. 1.3 The partially of O- and partially NH2-terminated diamond solution-gated field effect transistor (SGFET ) response of pH sensitivity. [23].

Diamond exhibits good sensing stability. Yang et al. have examined the denaturation process performance on diamond and a number of other materials including gold, silicon, gold

(16)

6

particles, and types of glassy carbon, thus demonstrating (Figure 1.4) that NCD is a high- stability substrate for selective biological activities [18].

Fig. 1.4 Stability of ultrananocrystalline diamond achieved for DNA-modified and other materials during 30 successive cycles of hybridization and denaturation [18].

From fig. 1.4, In each case, the substrates were amine-modified and then linked to thiol-terminated DNA using SSMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate) as a covalent linker [18]. After perfect hybridization from fluorophore labeled component, the intensity of fluorescence result are shown. Denaturation step was confirmed by intensity signal where fluorescence intensity become zero. [25].

Nanocrystalline diamond has acted as an effective biosensor for biological immobilization. The superiority of diamond is well-known, including its exceptional hardness, thermal conductivity, and biocompatibility (suitable for most biological environments), and the fact that it is stable and resistant from chemical attack [14]. The biosensor material requirements fulfilled by diamond are its surface functionalization, chemical inertness, mechanical shiftiness, optical transparency and electrical conductivity, which signifies the potential of diamond as a candidate for biosensor development [16].

(17)

7

1.1.2 Aptamers

Aptamers are single-stranded DNA, RNA, or peptide molecules that can bind a variety of chemical and biological molecules with high affinity and selectivity [26]. Aptamers are short nucleic acids that fold into complex three-dimensional structures and bind target molecules in a conformation-dependent manner. Aptamers have the potential to rival antibodies in many applications due to their high binding affinity and ability to discriminate between closely related targets. Furthermore, they can be very small (therefore reaching targets that may be inaccessible to antibodies) [27]. Due to their small size, aptamers are attractive for biosensor development with high stability (especially DNA aptamers), high binding affinity and specificity, and ease of modification. There are several reasons aptamers are very useful for biosensor applications:

 In principle, aptamers can be selected for any given target without soliciting immune response with low toxicity, and they have a little immunogenicity.

 The variety of functional groups of aptamers can be labeled at arbitrary positions, leading to high reproducibility of reagents well developed by chemical synthesis.

 The stability of aptamers is higher than that of proteins, and target binding can be repeated several times.

In 1990, Ellington and Szostack introduced that from in vitro selection RNA molecule that bind to specific ligand [28]. This introduction known as initial step, how aptamer began to develop into a very important part for development of technology, especially biosensor. In 1992, Ellington and Szostack continued to in vitro selection for single strand DNA and confirmed that Single strand DNA molecules fold into specific ligand binding structure [29].

From this invention, the special binding from in vitro process has grown rapidly become progressive part of aptamer development. Nowadays aptamer produced by systematic evolution of ligands by exponential enrichment (SELEX) process. SELEX process conducted through many cycles for optimizing affinity and specificity of aptamer which are chosen from huge random pool single stranded of nuclei acid sequences. These process can produce single

(18)

8

stranded aptamer in bases length around 15-60 (the longest one is 220 nucleotides) Figure 1.5 shows how SELEX process is conducted from a huge random sequence of oligonucleotides.

Fig. 1.5 Aptamer production by SELEX process.

Finally, due to their small size, aptamers may achieve a higher density for immobilization and bind to epitopes that are not easily accessed by antibodies. So far, the majority of the work has been carried out with DNA aptamers rather than RNA aptamers. In the previous concept, RNA aptamers used ATP and thrombin for binding ligands. RNA aptamers have high affinity binding and specificity for their target molecules. RNA aptamers directly bind with viral or specific ligands. RNA aptamers are used as a novel molecular technique, not as an aptasensor for ATP sensitivity detection, but as an investigation tool for a variety of viral and cellular proteins, as well as for development in diagnostics and as anti-viral agents. They have been used for viral selection as high affinity specific ligands for HCV, SARS, and HPV [27][30][32]. The incorporation of modified pyrimidine resulting in nuclease-resistant RNA aptamers makes them promising candidates for studying protein interactions in vitro and in vivo [33]. Aptamers have been explored as highly coveted molecular tools for both basic research and medical diagnosis regarding signaling expression for RNA aptamers. Signaling aptamers refer to aptamers or modified aptamers with recordable signal generation ability.

Through the SELEX process, many excellent RNA aptamers were obtained, as well as many riboswitches that can be potentially harnessed as biosensor components. RNA aptamers are

(19)

9

often obtained from reverse transcription, and it is difficult to label them. The challenging part of RNA aptamers is maintaining stability, which can be overcome by trapping them in assorted materials. RNA stability and binding affinity was improved by bases modification for many years. Biomedical diagnosis provides certain aptamer advantages over many other types of materials. For example, immobilizing aptamers in hydrogels might also be a good way to interfere with species [38].

The aptamer modification methods are required for rationally converting non-fluorescent aptamers into fluorescent aptamers directly from random-sequence DNA libraries by in vitro selection from standard DNA without inherently modifying fluorescence. The simple ways for aptamer signal generation process is needed for molecular recognition capability (no need for a difficult modification scheme to change an aptamer for signal generation). The fluorescence technique is highly compatible with nucleic acid aptamers.First, modification nucleic acids during the automated synthesis or by using simple post-synthesis chemistries are used fluorophore and quencher in a large. Second, fluorophore dressing of nucleic acids eliminates the need for target labeling and any aptamer–target pair it can be universally applied to all kind aptamer. Third, without the need for the separation of target– probe complexes from unbound probes and the detection can be carried out in real time can offered by fluorescence with very convenient way to report molecular interactions [30]. The main purpose of fluorescence detection is labeling process with many different fluorophores dye and quenchers, and also inherent for real time detection. Signaling aptamer was carried out in several strategies for converting aptamer into fluorescence signaling probe aptamer [31].

ATP molecule is capable being detected by selected aptamer in the µM range with contained only one fluorescence modified uredine. The signaling sensitivity and specificity could achieved in similar ways by fluorescence dyes [35].

The wide application of nucleic-acid aptamers in a range of areas has attracted intense interest.

Recent advances have been made in the development of aptamer-based biosensors and bioassay methods, most of which have been developed by electrochemical, optical, and mass- sensitive analytical techniques. DNA aptamers have high chemical stability [31]. DNA

(20)

10

aptamers do not need modifications for most applications [33]. The interaction of nonspecific electrostatic is easily to take place on a surface comes from DNA with highly negatively charged. The detection process was achieved by immobilization of DNA on a surface, the surface interaction between DNA, surface, and target in a sample is needed to understand from fundamental work [38]. DNA aptamers are easier to manipulate during the SELEX process and cheaper than RNA aptamers. Furthermore, the folded single strand DNA (ssDNA) has a less stability than RNA sequence. The folded structure of 3D configuration containing stem and loops. The conformations of DNA aptamers differ from the corresponding RNA aptamer sequences it also should be noted [37].

The DNA aptamers for ATP are the most common type of similar size (minimal functional structure of 32 and 25 nucleotides, respectively) and similar affinity and specificity for ATP.

Table 1.1 Comparison between aptamers and antibodies [32]

The advantages of aptamers are ease of modification and chemical synthesis, lower immunogenicity than antibodies, and the ability to refold after denaturation upon returning to the native condition. Aptamers are versatile biomaterials used as sensor elements, drugs, and drug delivery systems. The important view of high target affinity is a critical requirement for aptamers [26, 30-33].

(21)

11

ATP binding aptamer

ATP was first discovered by the German chemist Karl Lohmann [42], and the structure was established by Alexander Tood in 1948 [43]. The transferring of a phosphate group to other molecules was derived by ATP with endergonic reaction by phosphorylation. ATP is an important substrate in biological reactions and is known to be a key player in bioenergetics in biological systems [44]. ATP is the major carrier of chemical energy in living species and plays key roles in cellular metabolism and biological pathways in cell physiology. Adenosine is a component of many biological cofactors and combines with phosphate to form various chemical compounds. Label free ATP detection by fluorescence has been achieved by transfer of a labeled aptamer hybridized state to binding with ATP [30-34]. Adenosine is a component of many biological cofactors and combines with phosphate to form various chemical compounds, to assist in activity cell transport [36]. ATP contributes to mitochondria activity (respiratory) which result from reversible catalyzed reaction between ADP, ATP and AMP [44]. The reaction is shown below

2ADP = ATP + AMP [44].

(22)

12

When the phosphate group was added to adenosine diphosphate, the renewable result of ATP was generated. Figure 1.6 shows the chemical structure of ATP.

Fig. 1.6 Chemical structure of adenosine triphosphate [40]

As shown in Figure 1.6, ATP consist of purine base (adenine) attached to the first carbon atom of ribose (pentose sugar), and three phosphate groups are esterified at the fifth carbon atom on ribose. As the nucleotide world develops, new findings about indicated that they are interesting and challenging material for medical and sensing applications. In 1995, Huizenga first noted the ATP binding aptamer. The DNA ATP binding aptamer was first introduced by in vitro selection from a pool of ≈2x10¹⁴ different random sequence single stranded DNA molecules. The binding domain of the first ATP aptamer was localized to a 42 base sequence by deletion analysis. The G quartet on sequence and the two short items become active areas that are stacked with adenosine [39].

(23)

13

Fig. 1.7 Initial model of APT aptamer structure. This representation is not meant to suggest the exact orientation of any residue [39].

In 2003, Huang and Szostack successfully created the secondary structure of the ATP aptamer [41]. The mutation of a few bases resulted in changing the specificity of target detection, even without changing aptamer structure.

Fig. 1.8 First sequence of ATP aptamer and Watson-Crick pairing alignment for 27 mer ATP- binding DNA aptamer 1 identified through in vitro selection [39], (b) the present study for

(24)

14

the ATP binding 27-MEr DNA aptamer 1 complex determined in the secondary fold. With two nonequivalent AMP binding sites designated I and II in this complex [41]

Estrogen binding aptamer

Estrogen hormone has a similar structure to bisphenol A (BPA) which causes endocrine disrupting compounds such as reproductive disorder, chronic disease and various types of cancer [45]. Estrogen is natural steroid hormone that plays an important role in reproductive and sexual function. Estrogen plays a key role as a diagnostic marker of various chemical condition associated with sex hormone imbalance, such as puberty, fertility and ovarian tumors [46-49]. The 17-beta estradiol (E2) is a steroid hormone in the human body, is one of the most widely encountered endocrine disrupting chemicals in the environment [18,50-51]. E2 is even used as sin post menopause estrogen replacement therapy, making it one of the most encountered endocrine disrupting molecules in the environment. Endocrine disrupting molecules have caused cancerous tumors, birth defects, and development disorder in human, even in low concentration Natural and synthetic E2 have caused harmful effects in fish [2, 52-55]. E2 disruption has caused disequilibrium and dysfunction of the human immune system even in low concentration. Critical diseases such as breast cancer, ovarian cancer, and prostate cancer can occur from E2 explosion. E2 is the most active female sex hormone produced in ovaries that plays a crucial rule in the normal menstrual cycle [56-57].

Fig. 1.9 Structure of the 35-mer E2 aptamer and the 17β-estradiol (E2) molecule. Schematic illustration of the aptamer sensing process for E2 detection by the aptamer. (a) The E2

(25)

15

molecule (inset picture) as a target molecule for the E2 aptamer (not actual size). (b) The aptamer naturally forms a complex with the E2 molecule in the thymine-rich part [58].

The affinity of the estrogen aptamer occurred by the SELEX process leads high selection of aptamer which involves simple separation of DNA or RNA libraries. To recognize target molecules with the repetition of these steps will gradual increase in selection pressure. Aptamer affinity can be optimized by various conditions such as buffers, ions, pH, temperature, and environmental system. One of the important element of the aptamer binding ability of aptamers comprises hydrophilic oligonucleotides, and interaction between the aptamer and target involves hydrophobic ability, and very limited binding. Aptamer bind with their target with a part of the accessible surface area of their structure, and increase in the contact area between aptamers and targets would result in an increase in the binding affinity. Aptamer target binding is generally mediated by polar, hydrogen bonding, and charge-charge interaction. The circumstance of hydrophobic contact contributes to protein- interactions. The thermodynamically of Gibbs Free Energy (ΔG=-RTlnKa=ΔH-TΔS) is related to binding affinity of an aptamer. The entropic optimization can enhance the binding affinity maximally when aptamer target binding allow with the perfect fit, completely rigid with the multivalent construction. Lower Kd values result in a more sensitive sensor with a dynamic range. 35mer E2 aptamer was used to detect E2 target as low as 5nM in wastewater without any interference which is developed by optical sensor. [59, 60]. E2 binds to the loop formed by the thymine based (T-loop). A similar T-loop is presented in a circle where E2 molecule forms a complex with the aptamer [61].

1.2. Research Motivation

Diamond is promising material for merging solid and biological system. Using diamond as a biosensor has satisfied platform. The simplest diamond surface functionalize was done by attached biomolecule on diamond surface with chemical linker compound modification.

By surface functionalization, biological reactions, such as target recognition events, and associated sensing and signal processing can be integrated into a single platform. One of the

(26)

16

most important properties of diamond is its excellent chemical stability and reusability. In addition, oligonucleotides can be stably immobilized by covalent bonding with NH2 and COOH dangling bonds on the diamond surface. Hydrogen termination can be considered for grown diamond films and although diamond is composed of only carbons atom the surface stability achieved by bonding to element other than carbon. One of the potential advantages of diamond over alternative materials is that it provides extremely good stability and is fully compatible with microelectronic processing methods.

The uniqueness of biosensor designed using aptamer is the specific role as recognition elements. Aptamer have become the ideal recognition element for biosensor application.

Combining two element between aptamer and diamond is worth reporting and good combination for bimolecular reaction in the vicinity of functionalize carbon surface. There for we investigate aptamer for small molecule detection label free method on diamond surface via fluorescence signal detection.

1.3 Outline of the present Work

Aptamers have a key role in biosensor applications. Aptamer use has developed rapidly and they demonstrate varied functionality such as cancer cell detection, cancer theraphy, and biomolecule detection,. Aptamers are produced in vitro from single stranded nucleic acids or peptides with selective binding ability through a systematic evolution of ligands by exponential enrichment (SELEX) process using random sequences. The ability to bind with a variety of chemical and biological molecules with high affinity and selectivity is produced through repeating the SELEX process many times. The attractive qualities of aptamers for biosensor development include their small size, high stability, high binding affinity and specificity, and ease of modification, which is particularly applicable to DNA aptamers and RNA aptamers. Aptamers exhibit many advantages. First, in principle, they cannot producing significant toxicity without soliciting immune response for any given target. Second, reproducibility of aptamer is high and well developed by the chemical synthesis for labeled

(27)

17

at arbitrary position using a variety of functional groups. Third, aptamer stability is lead of proteins (compared with a traditional antibody) and can be repeatedly used without losing their binding capability. Fourth, they demonstrate little immunogenicity or toxicity. Finally, because of their small size, aptamers may achieve a higher density for attached DNA/RNA onto solid surface (immobilization) and bind to epitopes that are not easily accessed by antibodies. To date, most research has been carried out using DNA aptamers rather than RNA aptamers. Aptamers can be applied for simple methods and are ideal for the development of biosensors with high affinity, specificity, sensitivity, and stability. A label-free aptamer base, used for small molecule detection through fluorescence, was fabricated on nanocrystalline diamond (NCD). NCD demonstrates high chemical stability, simple functionalization, and biocompatibility. The simple surface functionalization was performed by attaching a biomolecule to the diamond surface using a chemical linker compound modification. Recent significant advances in chemistry may enable new aptamer combinations such as the biomolecular interaction of aptamers with chemically-functionalized surfaces such as those of diamond, gold nanoparticles, carbon nanotubes, and graphene. We expect that biomedical diagnostic, therapeutics, and bio analytics applications could be achieved by aptamers In this thesis, we report small molecule detection using a label-free technique using aptamers on diamond surfaces as a solid support. Diamond functionalization was conducted in advance in two parts, carboxyl termination and amine termination. Both terminations were conducted using a simple treatment. The proposed method is expected to provide a better and simple way for small molecule detection with high sensitivity, specificity, and reusability. This thesis consists of four chapters:

Chapter 1: INTRODUCTION. This chapter presents an overview of research trends in biosensors, with a focus on diamond-based carbon materials using aptamers as a biorecognition element. Aptamer biosensors represent a new type of sensor with unique properties. In many studies, diamond has been shown to possess high physical stability, a wide electrochemical potential window, chemical inertness, and biocompatibility. Diamond is a promising material in the production of biosensors and for merging solid and biological systems. The surface functionalization of solid materials is an important aspect of the

(28)

18

development of this field. Biological reactions, such as target recognition events, and associated sensing and signal processing can be integrated into a single platform through surface functionalization. Recently, one-step surface modification to directly produce amine groups on NCD for the patterning of oligonucleotides was conducted using a radical beam system. Radical treatment to functionalize diamond surfaces via amine termination is the latest dry treatment method for amine termination.

Chapter 2: SURFACE DEVELOPMENT FOR BIOMOLECULE IMMOBILIZATION. The importance of surface functionalization in maintaining stability and acting as a liaison between the diamond surface as a solid material and the aptamer as a biological material, and the importance of immobilization and blocking chemistry to reduce signal to noise or nonspecific binding for small molecule detection in real-word biological samples will be discussed. A diamond surface functionalization using carboxyl for hydrogen amine termination, amino groups for carboxyl termination, and fluorine termination is presented to improve the speed passivation layer of aptamer molecule interaction at the interface. In this chapter, amine termination via a nitrogen radical beam system is used to show the newest method to functionalize diamond surfaces for sensing applications.

Chapter 3: FLUORESCENCE SMALL NOLECULES DETECTION BY APTAMER.

This chapter is separated into several sub-chapters, which explore the ease of labeling aptamers with fluorescence dyes as a part of the sensing element and their inherent capability for faster real-time detection. The diamond was functionalized using different treatments and demonstrates significant potential as a sensing component. A proof of concept for aptasensing is explored in this chapter. The fluorescent dyes was used as sensing element which is part of labeled aptamers. The capability of real time detection situations for faster and simpler sensing of aptamers is discussed. Past research on fluorescence-based aptamer sensors is reviewed to define the progress in the development of aptamer-based sensors. Aptasensing with small molecule detection is explained well in previous studies with different methods and treatments. Past research is reviewed the aptamer applied into nanoparticle molecule for

(29)

19

ATP detection. In our work we used micro scale area, with fluorescence indicator directly immobilize and hybridize into diamond surface. The capability of surface functionalization for aptamer immobilization is an important requirement for aptasensing measurements. The aptamer label is based on the molecular light switch by CY-5 fluorophore molecule in a 5- end oligonucleotide modification. The reduction of fluorescence intensity confirmed as detection process.

3.1 ESTROGEN APTASENSOR. This chapter discusses the influence of diamond surface functionalization using a radical beam system for aptamers on the sensitivity of detection of low levels of estrogen hormone (E2/ 17-β estradiol). We investigate the sensitive and selective detection of E2 molecules in real-time using a fluorescence method on an amine terminated diamond surface. The E2 molecule is a steroid hormone which is present in the human body and various environmental media. This hormone is produced in the ovaries as the most active female sex hormone, and it plays a crucial rule in the normal menstrual cycle.

The dysregulation of this hormone, even at low levels, results in critical disease, including various types of cancer, and reproductive disorders. We focused on constructing an E2 sensing method using a fluorescence technique via aptamer binding capabilities. The simplest method, with high sensitivity and selectivity, is tried in a sensor device for use in realistic applications.

3.2 ATP APTASENSOR. Adenosine triphosphate (ATP) is an important substrate in biological reactions and is known to be a key player in bioenergetics in biological systems.

Adenosine is a component of many biological cofactors and combines with phosphate to form various chemical compounds. Label-free ATP detection using fluorescence has been achieved through the transfer of labeled aptamers to from a hybridized state with supporting DNA for to binding with ATP. The aptasensing strategy configuration was demonstrated for ATP detection using DNA-based aptamers and a fluorescence signal. Molecular switches of fluorescence intensity are a strategy for ATP detection. In the switch-off strategy, the method enables accurate detection of ATP excitation in the sample with a low signal to noise background based on physical absorption from terminated fluorine as a hydrophobic surface.

A detection limit of 1 nM was achieved. A different surface functionalization method was

(30)

20

demonstrated for ATP detection based on DNA aptamers. This subchapter is divided into three sub-sections.

3.2.1 Aptamer strategy for ATP detection on NCD functionalized using a nitrogen and hydrogen radical beam system

The nitrogen hydrogen radical beam system is one of the latest techniques for amine termination of solid surfaces. Diamond is a strong candidate for surface functionalization using this technique. This method is the simplest treatment for an amino terminated surface on a solid material. We functionalized a diamond surface for biosensing applications through partial oxidation and a nitrogen hydrogen termination process. Amine bonding was achieved using a radical source system by directly irradiating nitrogen and hydrogen radicals on the diamond surface. Nitrogen radical irradiation was carried out in a vacuum chamber for 20 min with a 200°C holder temperature. We detected ATP through the reduction of fluorescence from fluorescence dye labeled aptamer (DNA) when it forms a double stranded with complementary DNA (supporting DNA) immobilized on the diamond surface. The nitrogen coverage was estimated by integrating the photoelectron intensity of surface atoms, such as O 1s and N 1s, which are compared with the total photoelectron intensity of several layers of carbon. The mean was 0.96 Å. We obtained an average thickness of d = 0.561 Å for N 1s.

This means that N1s uniformly covered the diamond surface. Although not every nitrogen atom will be present as NH2, considering one monolayer of diamond, the maximum nitrogen group coverage estimated by the X-ray photoelectron spectroscopy (XPS) data is approximately 50%. From the maximum value of the monolayer, as much as 50% will be immobilized with single-stranded DNA. Aptamers will hybridize with supporting DNA depending on the concentration and hybridization density. A low concentration of nitrogen does not affect sensing ability. Above 0.5 Mono Layer (ML), the nitrogen coverage was suitable for biomolecule bonding with epifluorescence detection. Varying the concentration of supporting DNA used during the immobilization process and the aptamer concentration during the hybridization process can readily control the aptamer density on the surface.

3.2.2 The effect of nitrogen radical treatment and wet treatment for sensing application using aptamer. In this section, nitrogen termination using different techniques will be

(31)

21

explained briefly. Nitrogen hydrogen radical beam and ammonia solution treatments are surface functionalization techniques for amine termination. In a comparison with a chemical treatment using ammonia gas and an ammonia solution, the nitrogen radical beam (NRB) system was a good choice for safety, high compatibility, long-term storage and reusability. In addition, NRB was successful for Nitrogen Vacancy (NV) center growth near the surface.

Recently, NV centers and biomolecule centers are a hot issue in material science technology.

We investigate the characteristics of diamond that is partially aminated through direct amination using a nitrogen radical system and strongly immobilized supporting oligonucleotides to confirm the relationship between the aminated surface group and a supporting oligonucleotide modification with regards to hybridization. XPS and high resolution electron energy loss spectroscopy were used to determine the existence of nitrogen and other elements. The fluorescence intensity of the hybridized DNA was assessed using an epifluorescence microscope. Nitrogen radical exposure is safe with a diamond surface and the possibility of the physical absorption of nitrogen to the surface is extremely low.

3.2.3 Direct partial CH3 termination into diamond surface for biosensor. In this sub-chapter, CH3 partial termination for methyl terminal explained briefly. Washing treatment and stability of immobilization process was conducted in this chapter. The effect of CH3 terminals for carboxyl termination was explained briefly.

3.2.4 Aptamer-based carboxyl-terminated nanocrystalline diamond sensing arrays for adenosine triphosphate detection. In this sub-chapter, section carboxyl termination for ATP detection is explained briefly. Carboxyl termination is conducted using two treatment methods, vacuum ultraviolet (VUV) and ozone cleaner. Both VUV and ozone cleaner are conducted at room temperature and the average oxygen percentage of X-ray photoelectron spectroscopy (XPS) data after treatment is approximately ~1%. Above 1% carboxyl coverage was suitable for biomolecule bonding with epifluorescence detection. The dot pattern area on the diamond surface are rich of carboxyl terminal which was bonding with supporting DNA as immobilization process. Natural binding between two single strands of DNA forms double-stranded DNA. At this stage, the microscope captures the fluorescence signal. This fluorescence indicates successful hybridization between the supporting DNA and the DNA

(32)

22

aptamer with Cy5. In the second stage, ATP target release directly onto the diamond surface.

The double stranded DNA is disturbed by target. The aptamer released from the supporting DNA and forms a unique folding with ATP. This process demonstrates that aptamer has very specific binding and can detect a specific molecule. The ATP sensing detection recognized by reduction in the fluorescence of aptamer. This process occurred when aptamer partner changing from supporting DNA to ATP molecules. The stability of the sensor is shown by the fluorescence signals during the hybridization step. Fluorescence intensity variation is observed in seven cycles for 2 weeks. The ATP detection is sensitive until 1 nM, which is the lowest concentration used in this study. The intensity decrease depends on the ATP concentration.

Chapter 4: CONCLUSION AND FUTURE PERSPECTIVE. A brief summary of the major finding of this work and perspective on future directions on this project are given in this part.

The diamond surface functionalized for DNA aptamer sensing of small molecules is explained.

(33)

23

References

[1]. H. Kawarada, Hydrogen-terminated diamond surfaces and interfaces, Surface Science Reports, Volume 26, Issue 7, 205-259, ISSN 0167-5729 (1996)

[2]. S. Szunerits, C. Jama, Y. Coffinier, B. Marcus, D.Delabouglise, R. Boukherroub, Direct amination of hydrogen-terminated boron doped diamond surfaces, Electrochemistry Communications, Volume 8, Issue 7, 1185-1190, ISSN 1388-2481 (2006)

[3]. H. Kawarada and A. R. Ruslinda, Diamond electrolyte solution gate FETs for DNA and protein sensors using DNA/RNA aptamers” Phys. Status. Solidi A, 208（9）, 2005~2016 (2011)

[4]. T. Nakamura, T. Ohana, M. Hasegawa, Y. Koga, Chemical Modification of Diamond surface with fluorine containing functionalities. New Diamond and Frontier Carbon Technology. Vol. 15, No. 6 (2005)

[5]. S. Ferro and A. D. Battisti, "The 5-V Window of Polarizability of Fluorinated Diamond Electrodes in Aqueous Solutions,” Anal. Chem. 75, 7040 (2003)

[6]. J. H. Yang, S. Kuga, K. S. Song, H. Kawarada, "Characterization of Hybridization on Diamond Solution-Gate Field-Effect Transistors for Detecting Single Mismatched Oligonucleotides," Appl. Phys. Express, 1(11), 118001-1～118001-3, (2008).

[7]. T. Knickerbocker, T. Strother, M. P. Schwartz, J. N. Russell, J. Butler, L. M. smith, R.J.

Hamers, "DNA-Modified Diamond Surfaces," Langmuir, 19 (6), 1938–1942, (2003).

[8]. J. T. Paci, H. B. Man, B. Saha, D. Ho, G. C. Schatz, "Understanding the surfaces of nanodiamond," The J. of Phy. Chem. C. 33, 17256-17267, (2013).

[9]. X. Wang, Y. Ishii, A. R. Ruslinda, M. Hasegawa, H. Kawarada, "Effective Surface Functionalization of Nanocrystalline Diamond Films by Direct Carboxylation for PDGF Detection via Aptasensor, " ACS appl. Mat. & int., 4, 3526–3534, (2012).

(34)

24

[10]. E. Suaebah, T. Naramura, H. Kawarada, "Direct Partial CH3 Termination into Carboxyl Terminated Diamond Surface for Biosensor," IEEE proceeding, Busan, Korea, (2015).

[11]. J. H. Yang, K. S. Song, G. J. Zhang, M. Degawa, Y. Sasaki, I. Ohdomori, H. Kawarada,

"Characterization of DNA hybridization on partially aminated diamond by aromatic compounds," Langmuir, 22, 11245–11250, (2006).

[12]. S. Szunerits and R. Boukherroub, "Different strategies for functionalization of diamond surfaces, " J. of Solid State Elec.chem. 12, 1205–1218, (2008).

[13]. J. Hees*, R. Hoffmann, A. Kriele, W. Smirnov, H. Obloh, K. Glorer, B. Raynor, R.

Driad, N. Yang, O. A. Williams, and C. E. Nebel, Nanocrystalline Diamond Nanoelectrode Arrays and Ensembles, ACS Nano, , 5 (4), 3339–3346 (2011)

[14]. A. Härtl, E. Schmich, J. A. Garrido, J. Hernando, S. C. R. Catharino, S. Walter, P.

Feulner, A. Kromka, D. Steinmüller, M. Stutzmann, Protein-modified nanocrystalline diamond thin films for biosensor applications, Nature Materials 3, 736–742 (2004),

[15]. C. E. Nebel, D. Shin, B. Rezek, N. Tokuda, H.i Uetsuka, and H. Watanabe, Diamond and biology, J. R. Soc. Interface. 4(14): 439–461. (2007)

[16]. P. Ariano, O. Budnyk, S. Dalmazzo, D. Lovisolo, Ch. Manfredotti, P. Rivolo, E.

Vittone, On diamond surface properties and interactions with neurons, Eur. Phys. J. E. Soft Matter. Oct;30(2):149-56. (2009)

[17]. J. H. Yang, Y. Nakano, Y. Murakami, K. S. Song, H. Kawarada, Functionalization of ultra-dispersed diamond for DNA detection, Journal of Nanoparticle Research, 10, 69-75, (2008)

[18]. W. Yang, O. Auciello, J. E. Butler, W.Cai, J. A. Carlisle, J. E. Gerbi, D. M. Gruen, T.

Knickerbocker, T. L. Lasseter, J. N. Russell Jr., L. M. Smith, R. J. Hamers, DNA-modified nanocrystalline diamond thin-films as stable, biologically active substrates, Nature Materials 1, 253–257 (2002)

(35)

25

[19]. J. H. Yang, K. S. Song, S. Kuga, H. Kawarada, Characterization of Direct Immobilized Probe DNA on Partially Functionalized Diamond Solution-Gate Field-Effect Transistors, Japanese Journal of Applied Physics Vol. 45, No. 42, pp. L1114–L1117 (2006)

[20]. M. J. Song, S. K. Lee, J. Y. Lee, J. H. Kim, D. S. Lim, Electrochemical sensor based on Au nanoparticles decorated boron-doped diamond electrode using ferrocene-tagged aptamer for proton detection, J. of Electroanalytical Chem., Volumes 677–680, Pages 139- 144, ISSN 1572-6657 (2012)

[21]. A. Härtl, E. Schmich, J. A. Garrido, J. Hernando, S. C. R. Catharino, S. Walter, P.

Feulner, A. Kromka, D. Steinmüller& M. Stutzmann, Protein-modified nanocrystalline diamond thin films for biosensor applications, Nature Materials 3, 736–742 (2004)

[22] B. Rezek,D. Shin, and C. E. Nebel, Properties of Hybridized DNA Arrays on Single- Crystalline Undoped and Boron-Doped (100) Diamonds Studied by Atomic Force Microscopy in Electrolytes, Langmuir, 23 (14), pp 7626–7633 (2007)

[23]. Y. Sasaki and H. Kawarada, Low drift and small hysteresis characteristics of diamond electrolyte-solution-gate FET, Journal of Physics D: Applied Physics, Volume 43, Number 37 (2010)

[24] V. Vermeeren, N. Bijnens, L. Grieten, S. Wenmackers, N. Vanden Bon, K. Haenen, P.

Wagner, L. Michiels Diamond-based Biosensors with an Impedimetric and Label-free Read- out, microtech conference and expo (2010)

[25]. Strother, T. et al. Photochemical functionalization of diamond films. Langmuir 18, 968–

971 (2002).

[26]. M. V. Yigit, D. Mazumdar, H.-K. Kim, J. H. Lee, B. Odintsov, and Y. Lu, “Smart ‘turn- on’ magnetic resonance contrast agents based on aptamer-functionalized superparamagnetic iron oxide nanoparticles.,” Chembiochem, vol. 8, no. 14, pp. 1675–8 (2007)

(36)

26

[27] C. Nicol, Ö. Cesur, S. Forrest, T. a Belyaeva, D. H. J. Bunka, G. E. Blair, and N. J.

Stonehouse, “An RNA aptamer provides a novel approach for the induction of apoptosis by targeting the HPV16 E7 oncoprotein.,” PLoS One, vol. 8, no. 5, p. e64781, (2013)

[28]. A. D. Ellington & J. W. Szostak, In vitro selection of RNA molecules that bind specific ligands, Nature, 346, 818–822 (1990)

[29]. A. D. Ellington & J. W. Szostak, Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures, Nature, 355, 850–852 (1992)

[30]. R. Nutiu and Y. Li, “Aptamers with fluorescence-signaling properties.” Methods, vol.

37, no. 1, pp. 16–25, (2005)

[31]. S. Song, L. Wang, J. Li, J. Zhao, and C. Fan, “Aptamer-based biosensors,” vol. 27, no.

2, pp. 108–117, (2008)

[32]. K. Kang and Y. Lee, “RNA Aptamers : A Review of Recent Trends and Applications,”

Adv Biochem Eng Biotecnol, vol. 131, no. 2013, pp. 153–169 (2013)

[33]. H. Ulrich, A. H. B. Martins, and J. B. Pesquero, “RNA and DNA aptamers in cytomics analysis.,” Curr. Protoc. Cytom., vol. Chapter 7, p. Unit 7.28, (2005)

[34]. R. R. Breaker, “DNA aptamers and DNA enzymes,” pp. 26–31, (1997)

[35]. T. G. Rongsheng, E. Wang, Y. Zhang, J. Cai, W. Cai, “APtamer based Fluorescent Biosensor,” vol. 18, no. 668, pp. 4175–4184, (2011)

[36] D. J. P. U and A. K. Suri, “Structure, recognition and discrimination in RNA aptamer complexes with cofactors , amino acids , drugs and aminoglycoside antibiotics,(2000) [37] F. W. S. Amine, A. Yarman, T. Bachmann, T. Hirsch, S. Kubick, R. Renneberg, S.

Schumacher, U. Wollenberger,et. al, Biosensors Based on Aptamers and Enzymes, vol. 140.

Berlin, Heidelberg: Springer Berlin Heidelberg, p. 338 (2014)

[38]. W. Zhou, P.-J. Jimmy Huang, J. Ding, and J. Liu, “Aptamer-based biosensors for biomedical diagnostics.,” Analyst, (2014)

(37)

27

[39]. D. E. Huizenga and J. W. Szostak, “A DNA Aptamer That Binds Adenosine and ATP,”

Biochemistry, vol. 34, pp. 656–665, (1995)

[40].((https://pubchem.ncbi.nlm.nih.gov/compound/Adenosine_triphosphate#section=Top)) accessed 20 December 2017

[41]. C. H. Lin, D. J. Patei, Structural basis of DNA folding and recognition in an AMP- DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP, Chemistry & Biology, Volume 4, 11, , P 817-832, (1997) [42]. P. Langen, F. Hucho, Karl Lohmann and the Discovery of ATP, (2008)

[43]. M.A. Shampo, R. A. Kyle, and D. P. Steensmab, Alexander Todd—British Nobel Laureate, Mayo Clin Proc. Mar; 87(3): e19. (2012)

[44]. https://pubchem.ncbi.nlm.nih.gov/compound/Adenosine_triphosphate, accessed 20 December 2017.

[45]. V. Belgiorno, L. Rizzo, D. Fatta, C. D. Rocca, G. Lofrano, A. Nikolaou, V. Naddeo, Sureyya Meric, Review on endocrine disrupting-emerging compounds in urban wastewater:

occurrence and removal by photocatalysis and ultrasonic irradiation for wastewater reuse, Desalination, Volume 215, Issues 1–3 (2007)

[46]. M. Sun, L. Du, S. Gao, Y. Bao, S. Wang, Determination of 17β-oestradiol by fluorescence immunoassay with streptavidin-conjugated quantum dots as label, In Steroids, Volume 75, Issue 6, , Pages 400-403 (2010)

[47]. G.G. Ying, R.S. Kookana, Y.J. Ru, Occurrence and fate of hormone steroids in the environment, Environ. Int. 28 545–551. (2002)

[48]. V. N. Mochalin, O. Shenderova, D. Ho, & Y. Gogotsi, The properties and applications of nanodiamonds. Nat. Nanotechnol. 7, 11–23 (2011).

[49]. M. Famulok, & G. Mayer, Aptamer modules as sensors and detectors. Acc. Chem. Res.

44, 1349–58 (2011).

(38)

28

[50]. G. S. Baird, Where are all the aptamers? Am. J. Clin. Pathol. 134, 529–31 (2010).

[51]. H. Kuramitz, M. Matsuda, J. H. Thomas, K. Sugawarac and S. Tanaka, Electrochemical immunoassay at a 17β-estradiol self-assembled monolayer electrode using a redox marker, analyst (2003)

[52]. G.G. Ying, R.S. Kookana, Y.J. Ru, Occurrence and fate of hormone steroids in the environment, Environ. Int. 28 545–551(2002)

[53].B. Zhu, O.A. Alsager, S. Kumar, J.M. Hodgkiss, J. Travas-Sejdic, Label-free electrochemical aptasensor for femtomolar detection of 17β-estradiol, Biosens. Bioelectron.

70 398–403 (2015).

[54]. O. A. Alsager, S. Kumar, B. Zhu, J. T.Sejdic, K. P. McNatty, and J. M. Hodgkiss, Ultrasensitive Colorimetric Detection of 17β-Estradiol: The Effect of Shortening DNA Aptamer Sequences, Analytical Chemistry 87 (8), 4201-4209 (2015)

[55]. P., Iv, E. Wagner, & L. Sept, Diamond-based biosensors for nucleic acids, proteins and drugs. (2008).

[56]. S. G. Kim, J. S. Lee, J. Jun, D. H. Shin, & J. Jang, Ultrasensitive Bisphenol A Field- Effect Transistor Sensor Using an Aptamer-Modified Multichannel Carbon Nanofiber Transducer. ACS Appl. Mater. Interfaces 8, 6602–6610 (2016).

[57]. H. Y. Zheng, O. A. Alsager, C. S. Wood, J. M. Hodgkiss, N. O. V. Plank, Carbon nanotube field effect transistor aptasensors for estrogen detection in liquids. J. Vac. Sci.

Technol. B, Nanotechnol. Microelectron. Mater. Process. Meas. Phenom. 33, 06F904 (2015).

[58]. N. Yildirim, et al. Aptamer-Based Optical Biosensor For Rapid and Sensitive Detection of 17 β -Estradiol In Water Samples. (2012).

[59]. S. U. Akki, C. J. Werth, & S. K. Silverman, Selective Aptamers for Detection of Estradiol and Ethynylestradiol in Natural Waters. (2015).

(39)

29

[60]. H. Hasegawa, N. Savory, K. Abe,& K. Ikebukuro, Methods for Improving Aptamer Binding Affinity. (2016).

[61]. T. A. Hilder, & J. M. Hodgkiss, The Bound Structures of 17 b -Estradiol-Binding Aptamers. 1881–1887 (2017).

(40)

30

Chapter 2 Surface development for

biomolecule immobilization

(41)

31

2.1 Introduction

Many methods are used for diamond surface functionalization including plasma, wet process, chemical process, and dry process [1-4]. The diamond surface is terminated with several functionalization elements such as hydrogen, oxygen, carboxyl, amine, and fluorine.

For transistor application, hydrogen and oxygen can be applied for surface functionalization.

For biomolecule immobilization, carboxyl and amine termination can be applied very well, and fluorine termination can be applied for nonspecific bonding adsorption in the immobilization technique (Fig 2.1).

Fig. 2.1 Typical surface functionalization achieved directly on diamond surface [5]

Due to differences in the surface functionalization, electronegativity may be induced in significant different from diamond materials. In the following part, four types of typical termination will be compared.

2.2 Hydrogen Termination

A hydrogen-terminated surface is induced via hydrogen plasma in the chemical vapor deposition (CVD) system. Hydrogen plasma benefits the diamond surface by removing

(42)

32

chemical contamination and all other types of surface termination. This is very useful for recycling applications for diamond substrate [6].

Fig. 2.2 Hydrogenated by plasma treatment on diamond surface.

Without hydrogen termination, a diamond surface with a sp3 dangling bond becomes inactive and unstable. To improve electronegativity for semiconductor application, hydrogen and carbon bonding achieved by hydrogen termination provides stable and effective C-H bonding in air or metal deposition. The surface state is effectively reduced by the hydrogen termination [7]. The surface conductivity of hydrogen is improved by hydrogen termination [8]. Standard atmospheric conditions will not result in hole accumulation within those materials. Hydrogenated treatment increases the valence band maximum (VBM) of diamonds sufficiently. Electron transfer from the diamond to the H3O⁺/ (H2O+H2) redox couple accounts for the hole accumulation layer. The Fermi level of a diamond after hydrogenation is pinned onto up level, which is coincident essentially with the VBM [8].

Spontaneous polarization of hydrogen termination has a positive charge by C-H dipole. When the surface is exposed to the air, negatively charged ions are captured by the positively charged surface. The negatively charged ions induce upward-band-bending on the diamond surface [7]. Additionally, holes accumulate on the diamond surface to form 2-dimensional hole gas. The hydrogenated diamond surface has several advantages. It can display p-type conductivity, has a stabilizing surface structure, has a wide band gap, exhibits high thermal conductivity, eliminates surface dangling bonds, and reduces the number of defects [9-11].

(43)

33

Fig. 2.3 Schematic diagrams of functionalization conversion from di- and tri-hydrogen termination to carboxyl groups on diamond [2].

To produced hydrogen termination, a nanocrystalline diamond (NCD) was exposed to methane containing plasma to realize partial CH3 termination (Fig. 2.2). This was performed using hydrogen gas diluted with methane gas via a microwave plasma chemical vapor deposition (MVCVD) system with a chamber pressure. After 30 min, partial CH3 termination had occurred (less than 10%); both gases were stopped, and 1 hour was allotted for the cooling process. Hydrogen termination is an initial process to improve the surface characteristics of diamond. The hydrogen-terminated surface was the starting surface before nitrogen/hydrogen radical irradiation onto the diamond surface. The hydrogen-terminated surface can coexist with the nitrogen-terminated surface.

(44)

34

2.3 Carboxyl Termination

Carboxyl termination is conducted to produce COOH bonding on a diamond surface.

Carboxyl termination is one choice to functionalize a diamond surface via oxidation.

Oxidation is relatively easy to perform [12] and relatively stable by environmental conditions [13-14]. There are many ways to produce carboxyl termination on a diamond surface; firstly, vacuum ultraviolet (VUV) treatment was introduced in 2004 by Ohta [15]. In the same year, ozone treatment was used by Kawarada to produce carboxyl termination on a diamond surface [16]. Another method to produce carboxyl termination is by plasma treatment [17].

Fig. 2.4 Top: carboxyl termination schematic chamber by VUV chamber and ozone cleaner chamber. Bottom: surface functionalization C-H bonding after hydrogen termination becomes oxidized.

Subsequently, carboxyl modification was conducted using the dry technique. UV ozone cleaner and VUV treatment were used to produce carboxyl termination on the diamond surface. The resulting carboxyl group on the diamond surface was used to covalently attach amino-labeled DNA. To partially CH3 terminate the NCD surface, NCD on silicon was exposed to methane-containing (3%) plasma (297 sccm hydrogen gas diluted with 3 sccm methane gas) for 30 min using an MVCVD system with a chamber pressure of 50 Torr, power

(45)

35

of 1.2 kW, and temperature of ~700 °C. After 30 min, partial CH3 termination was achieved, the flow of both gases was stopped, and the reaction system was cooled for 1 h.

Direct partial carboxyl termination was performed on the partially CH3-terminated NCD surface. NCD was functionalized by UV irradiation to directly produce carboxyl groups. UV irradiation was performed using a Xenon excimer lamp with a lamp power of 20 W to generate an ultraviolet laser with a wavelength of 172 nm. Pure oxygen was introduced into the chamber to achieve a pressure of 3 × 10⁴ Pa, and then UV irradiation was performed for 45 min at room temperature. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface chemical change after carboxyl termination. XPS electron spectrometry was conducted using an Ulvac Φ 3300 (Ulvac-Phi, Kanagawa, Japan) with a monochromatic Al Kα X-ray source.

A micropattern was fabricated on the NCD surface by photolithography. Gold (150 nm thick) was deposited on the NCD surface. Before patterning by an aligner, the NCD surface was pre-baked for 20 min at 80 °C to remove all water molecules. The gold layer was coated with a resist film by spin coating, aged for 20 min at room temperature, and then baked at 100 °C for 5 min. The sample was then patterned with the aligner for 33 s. The negative pattern produced by photolithography was obtained by gold etching with KI/I2 etching solution. The outside of the dot pattern was exposed to C3F8 plasma (RIE-101iPH; Samco International Inc., Tokyo, Japan) for 15 s to generate a fluorine-terminated surface as a passivated layer outside the dot pattern. This process minimizes nonspecific adsorption of supporting molecules and the aptamer. A schematic diagram of the fabrication of the micropatterned NCD surface is shown in Figure 2.5.

(46)

36

Fig. 2.5 Schematic diagram of micropattern fabrication on the nanocrystalline diamond (NCD) surface. (a) Hydrogen termination; (b) Vacuum ultraviolet (VUV)/Ozone treatment;

(c) Gold deposition; (d) Photolithography process; (e) Fluorine termination; (f) Au mask etching; (g) Optical microscopy image of the dot pattern formed on the NCD surface. The dots are terminated by carboxyl groups, and regions outside the dots are fluorine-terminated as a background.

Figure 2.5 shows a schematic diagram of NCD functionalization to produce the carboxyl- terminated surface. Micropatterning produced a dot pattern with 20 μm diameter dots separated by a distance of 20 μm. The dot patterns are partially carboxyl-terminated, and the fluorinated surface is the background. An optical microscopy image of the patterned NCD surface covered by gold before final etching with carboxyl- and fluorine-terminated regions is shown in Figure 2.5 (g).

Final etching was performed to remove the gold area on the NCD, and the remaining area covered with gold was carboxyl terminated. The supporting DNA was immobilized on the carboxyl-terminated dots after the final etching process. Carboxyl termination enables covalent immobilization of supporting DNA, which hybridizes with the aptamer for

Functionalized diamond surfaces for DNA aptamer sensing of small molecules

Functionalized diamond surfaces for DNA aptamer sensing of small

molecules

February 2018

EVI Suaebah

スアエバ エヴィ

Functionalized diamond surfaces for DNA aptamer sensing of small

molecules

February 2018

Waseda University

Graduate School of Advance Science and Engineering Department of Nanoscience and Nanoengineering

Research on Nanodevices EVI Suaebah

スアエバ エヴィ

Dedication

Acknowledgement

Abstract

Contents

Chapter 1

Introduction

1.1 Research Background 1.1.1 Diamond Biosensor

1.1.2 Aptamers

ATP binding aptamer

Estrogen binding aptamer

1.2. Research Motivation

1.3 Outline of the present Work

References

Chapter 2

Surface development for

biomolecule immobilization

2.1 Introduction

2.2 Hydrogen Termination

2.3 Carboxyl Termination

スアエバエヴィ

スアエバエヴィ