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 photostability tests of nanodiamond (red) and fluorescent polystyrene nanospheres
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(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 chips. The biochip can be able to large numbers of biological analytes quickly for
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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.
The signals such as variations of electric or optical properties were used to take as
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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.
The acoustic cavitation [3.8, 3.9] generated by ultrasonic bath heats up the water to
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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 dimensional organization of hydrophobic tail groups. Initially, adsorbate molecules
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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 carboxylated NDs were anchored on the patterned silicon templates and processes for
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the preparation of the substrates.
3-2-3 Fabrication of PLL and FITC functionalized nanodiamond nanoarray
The PLL (poly-L-lysine hydrobromide) solution was prepared by dissolving 8ml PLL into 20ml borate buffer and the FITC (fluorescence isothiocyanate) solution was prepared by dissolving into 10mg FITC into 3ml borate buffer. The nanodiamond array chip was dipped into the 3ml borate buffer and reacted with the added 1ml PLL solution for 60 minutes. After reaction, the nanodiamond array chip was washed by borate buffer and deionized water. The PLL functionalized nanodiamond arrays were demonstrated. The adsorption of PLL on the nanodiamond arrays was further verified using the FTIC as a probe. Then, the chip was dipped into the mixture of 3ml borate buffer and 0.02ml FITC solution and reacted for 30 minutes in dark room. The superfluous FITC was washed by DMSO, borate buffer and deionzed water. All of the processes were carried under 4 oC. The figure 3-6 shows the processes of the nanodiamond array chip which was functionalized by PLL and attached with FITC.
[3.11]
3-2-4 Fabrication of Lysozyme functionalized nanodiamond arrays
The lysozyme solution was prepared by dissolving 0.1g lysozyme into 20ml PBS (Phosphate-buffered saline) buffer. To ensure equilibration absorption, the
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nanodiamond patterned chip was dipped into the lysozyme solution mentioned above and mixed together with stirring for 2hr before it was washed by PBS buffer and deionized water. After which, 10μl of E.coli suspension in 90μl PBS medium was mixed with the nanodiamonds chip in PBS buffer. The nanodiamond chip was washed with PBS buffer and deionized water. The lysozyme fuctionalized nanodiamond array was preserved under 4oC before examination.
3-3 Results and discussion
3-3-1 Carboxylated nanodiamond
The figure 3-7 (a) and (b) show the Raman and PL spectra of NDs with and without the acid treatment at an excitation wavelength of 488 nm. The treatment with acid particularly removed the carbon-like structure from the ND surface. In the Raman spectrum, the peaks at 1350 cm-1 and 1580 cm-1 (D-band and G-band signals caused by the carbon-like SP2 structure from the ND surface) were clearly attenuated after the acid treatment [3.12]. The figure 3-7(b) shows that the narrower emission band was obtained because the acid treatment reduced the surface disorder and the number of surface defects [3.13]. The figure 3-8 shows the Fourier transform infrared (FTIR) spectroscopy of NDs before and after acid treatment. In the FTIR spectrum, the
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surface carboxylated groups induce the peaks between 3100 and 3300 cm−1 broadening [3.7].
3-3-2 Nanodimond nanoarray
Two kinds of patterns were designed to be placed on the Si templates. One is the
crossmarks and the other one is nanohole arrays. The crossmarks have a length of 600 μm and a width of 20 μm. The nanohole arrays have an average diameter of 300 nm
and a pitch size of 5 μm. The figure 3-9 (a) shows the scanning electron microscope (SEM) image of one of the corners inside the crossmark. The SEM image of 2 dimensional nano arrays with a diameter of 300 nm and the pitch of the array in 5 μm is shown in Figure 3-9(b). From the SEM images one can observe that the nanodiamonds bonded with the patterned SAM inside the nano holes. The inset in Figure 3-9(b) shows a single functionalized ND was isolated inside a nano hole. The SEM image proves that we were able to arrange NDs with various well-defined cross-section SEM image and the AFM image, the well patterned single nanodimaond
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array can be proved.
The optical properties of the patterned nanodiamonds are demonstrated as follows.
The micro-Raman spectra were also excited inside the reference crossmarks, nano holes, and outside the nano hole array with a laser beam of about 1 μm in diameter.
The Raman signals, as shown in Figure 3-11(a), were only found inside the crossmarks and nano holes array where the NDs were anchored. However, with the laser beam placed outside the nano hole array (pattern-out area), no diamond-related signals were collected. This indicates that NDs were only allocated on the SAM inside the crossmarks and the nano hole arrays. The locations of the anchored NDs were further examined with the photoluminescence spectra excited at a wavelength of 532 nm. The PL spectra are shown in Figure 3-11(b). The PL spectrum of the ND clusters is also shown in Figure 3-11(b) for comparison. Again, no PL signals can be found outside the pattern area.
A one dimensional (1D) Raman intensity image mapping was carried out along a selected nano hole array, as shown in figure 3-12(a). The laser beam with a diameter of 1μm was scanned along a distance of 20μm with a 500 nm step. The intensity of
the 1332 cm-1 Raman signal as a function of the scanning distance was plotted in figure 3-12(b). We also performed a two-dimensional (2D) Raman spectra mapping in a selected square area (the area is shown by the red square in figure 3-13(a). The 2D
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image of integrated Raman intensity mapping of the 1332cm-1 Raman peak is shown
in figure 3-13(b). Keep in mind that the nanodiamond arrays were designed with a pitch of 5μm. When compared with the results from the 2D Raman intensity mapping,
we found that the intensity distribution was perfectly correlated with the spatial distribution of the nano hole arrays. The results indicate that the NDs were only anchored inside the nano holes and were perfectly distributed on the template according to the pattern defined by the e-beam lithography technique.
3-3-3 PLL and FITC functionalized nanodiamond arrays.
In the figure 3-14(a) shows the optical microscope image of the PLL and FITC functionalize nanodiamond arrays. The FICT dye was excited by 488nm laser and emission at 518nm. The 1D PL mapping was demonstrated along the selected red line in the figure 3-12(a). The results of PL mapping shown in figure 3-14(b) indicates that the intensity distribution of FITC was perfectly correlated with the spatial distribution of the PLL and FITC functionalized nanodiamond arrays. It was proved by detecting the PL signals from FITC that the nanodiamond arrays can be successfully functionalized by PLL. It was proved that the nanodiamond arrays can be functionalized by bio-molecules under above examination.
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3-3-4 Lysozyme functionalized nanodiamond arrays
The lysozyme solution was prepared by dissolving 0.1g lysozyme into 20ml PBS (Phosphate-buffered saline) buffer. To ensure equilibration absorption, the nanodiamond patterned chip was dipped into the lysozyme solution mentioned above
and mixed together with stirring for 2hr before it was washed by PBS buffer and deionized water. After which, 10μl of E.coli suspension in 90μl PBS medium was
mixed with the nanodiamonds chip in PBS buffer. The nanodiamond chip was washed with PBS buffer and deionized water. The lysozyme fuctionalized nanodiamond array was preserved under 4oC before examination.
In the figure 3-15, the FTIR (Fourier transform infrared) spectra were shown for three different samples of carboxylated nanodiamond, lysozyme protein, and lysozyme functionalized nanodiamond arrays [3.7]. The carboxylated nanodiamonds show the peaks of C=O group at 1750cm-1and of carboxylated group at 3000cm-1-3500cm-1. The ratio of carboxylated groups on nanodimond surface is about 7% [3.14, 3.15]. It makes the weaker signal of the surface carboxylated groups but the carboxylated group of nanodiamonds surface can be confirmed in FTIR spectrum.
The FTIR spectrum of lysozyme is shown in Figure 3-15(b). The appearance of amide peaks is at 1490 cm-1 - 1590 cm-1 (amide 1), 1600 cm-1 - 1700 cm-1 (amide 2), and 3100 cm-1 - 3300 cm-1 from the lysozyme [3.7]. The spectra of lysozyme
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functionalized nanodiamond arrays are shown Figure 3-15(c). Due to the large background from the SiO2 layer for the energy larger than 3000 cm-1, detecting any peaks higher than 3000 cm-1 is difficult. However, weak peaks of amide at 1490 cm-1 - 1590 cm-1 and 1600 cm-1 - 1700 cm-1 that from lysozyme can still be identified, as shown in Figure 3-15(c).
The investigation of Raman spectra for the three different samples of lysozyme, lysozyme functionalized nanodiamond solution, and lysozyme functionalized nanodiamond arrays are shown in Figure 3-16. The figure 3-16(a) shows the Raman spectrum of the protein lysozyme. In the region between 1400 cm-1 and 1700 cm-1, some weak peaks were found due to amide in protein, amino acid, CH, and CH2 groups. Figures 3-16(b) and 3-16(c) show the Raman spectra of lysozyme functionalized nanodiamond solution and lysozyme functionalized nanodiamond arrays, respectively. As shown in the spectra, the functionalized nanodiamond solution exhibits both peaks of nanodiamond located at 1332 cm-1 and lysozyme located at the region between 1400 cm-1 and 1700 cm-1. Within our expectation, the Raman spectrum of the NDs-lysozyme arrays on the silicon template is identical to the NDs-lysozyme complex in the solution. After placement of lysozyme functionalized nanodiamond arrays in room temperate for 24 hours, the Raman spectrum of lysozyme functionalized nanodiamond arrays (don’t show here) is unidentifiable for
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lysozyme Raman feature. It may interpret the activity of lysozyme functionalized nanodiamond arrays vanish under such condition.
The interaction of the stable and bioactive lysozyme functionalized nanodiamond arrays with bacteria can be verified and observed using SEM. For the observation of interaction between lysozyme functionalized nanodiamond arrays and E.coli, the nanodiamond arrays were designed into 1μm in length and 5μm in pitch. Figures 3-17 (a) and (b) show the SEM images that E.coli interaction with the lysozyme functionalized nanodiamond crossmarks and nanoarrays, respectively. Form the SEM images, we clearly observed that the E.coli were morphologically damaged while absorbed on lysozyme functionalized nanodiamond arrays [3.16, 3.17]. Lysozyme is an enzyme, which hydrolyze the cell wall of the bacteria. The lysozyme proteins absorbed on the nanodiamond arrays still retained their antibacterial activity and interacted with the E.coli bacterial cells. As shown in the figure 3-17 (b), the E.coli only interacts with the lysozyme functionalized nanodiamond arrays. It can’t be be observed that E.coli interact with the silicon substrate without lysozyme functionalized nanodiamond arrays.
3-4 Summary
In this study, we have demonstrated new methods and techniques to anchor
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bio-functionalized nanodiamond arrays on silicon template by using e-beam lithography and SAM techniques. The nanodiamond arrays can be functionalized by kinds of bio-molecules such as PLL and lysozyme proteins. The lysozyme proteins can functionalize the nanodiamonds and still retained their antibacterial activity and interacted with E. coli bacterial cells. The device demonstrated here is suitable for applications in bio-sensing chips and single bio-molecule patterning and detection. It facilitates the development of new applications of different bio-functionalized nanodiamond arrays that can interact with special targets, as well as the individual observation of their optical property.
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