國 立 交 通 大 學
生 物 科 技 學 院
生 物 科 技 學 系
碩 士 論 文
建構一以奈米金球為基礎的光學生物感測平台用於
蛋白質酶活性之檢測
Establishing a Gold Nanoparticles-Based Optical Biosensing
Platform for the Assay of Proteinase Activity
研 究 生：李榕均
指導教授：林志生 博士
建構一以奈米金球為基礎的光學生物感測平台用於蛋白質酶
活性之檢測
Establishing a gold nanoparticles-based optical biosensing
platform for the assay of proteinase activity
研 究 生：李榕均 Student：Jung-Chun Li
指導教授：林志生 Advisor：Chih-Sheng Lin
國 立 交 通 大 學
生 物 科 技 學 院
生 物 科 技 學 系
碩 士 論 文
A ThesisSubmitted to Department of Biological Science and Technology College of Biological Science and Technology
National Chiao Tung University In partial Fulfillment of the Requirements
For the Degree of Master In
Biological Science and Technology
July 2009
Hsinchu, Taiwan, Republic of China
建構一以奈米金球為基礎的光學生物感測平台用於蛋白質酶
活性之檢測
研究生：李榕均 指導教授：林志生 博士
國 立 交 通 大 學
生 物 科 技 學 院
生 物 科 技 學 系
摘 要
奈米金球（gold nanoparticles, AuNPs）具有獨特的物理特性，並且在光學上具有表 面電漿共振吸收（surface plasmon resonance, SPR）的現象而備受注目。由於奈米金球的
吸收光譜會因大小、形狀，或表面修飾上有機分子而有所差異，因此本研究利用AuNPs
之SPR 特性，建構出一光學式生物感測平台用於蛋白質酶（proteinase）活性之檢測。
本研究首先將明膠（gelatin）修飾於 13 nm 的 AuNPs 上作為 proteinase 之受質，同 時修飾上6-醯基己-1-醇（6-mercapto-1-hexanol, MCH）當作誘導子。當 AuNPs 修飾上 gelatin 與 MCH 時，gelatin 所造成的空間障礙可防止金球彼此間的距離拉近，避免 AuNPs 產生聚集。因此AuNPs/MCH-gelatin 可穩定存在於極端的檢測環境中。當 proteinase 中 的胰蛋白酶（trypsin）或基質金屬蛋白質酶-2（matrix metalloproteinase-2, MMP-2）降解 AuNPs/MCH-gelatin 表面上的受質後，AuNPs 失去保護，同時 MCH 增強 AuNPs 彼此間 的吸引力，AuNPs 彼此間距離逐漸靠近，因而產生聚集。AuNPs 聚集時，其 SPR 會有 紅位移（red-shift）現象，並且 AuNPs 的呈色會由原先的酒紅色轉變為紫色。此外，AuNPs 之顏色變化可直接以肉眼觀察，且最大吸收波峰值（λmax）可經由UV/Vis spectroscopy 量測之。
在此以奈米金球為基礎的光學式生物感測平台中，利用 AuNPs 的吸光值比值 （A625 nm/A525 nm）估計proteinase 之活性，針對 trypsin 之偵測線性範圍為5 × 10-1至5 × 102 U，偵測極限為 5 × 10-1 U；而針對 MMP-2測得的線性範圍為50 ng/mL 到 600 ng/mL， 偵測極限為50 ng/mL。此外利用兩種 MMP-2 抑制劑，galardin 及 ONO-4817 進行藥物篩 檢方面研究。利用此奈米金球系統針對galardin 及 ONO-48 進行檢測，其 IC50值分別為 1.87 nM 以及 17.76 nM，而以酶譜分析法（zymography）所測得之 IC50值分別為3.48 與14.33 nM，結果顯示此兩種方式所測得之結果有高度一致性。然而此光學式生物感測 系統可將偵測時間縮短到30 分鐘內。因此，此快速且敏感性高的奈米金球光學生物感 測平台具有相當潛力運用於anti-MMPs 藥物篩檢及 MMPs 相關疾病診斷上。 關鍵字：奈米金球，蛋白質酶，光學式生物感測平台，基質金屬蛋白酶，明膠
Establishing a gold nanoparticles-based optical biosensing
platform for the assay of proteinase activity
Graduate student: Jung-Chun Li Advisor: Chih-Sheng Lin Ph. D.
Department of Biological Science and Technology
College of Biological Science and Technology
National Chiao Tung University
Abstract
Gold nanoparticles (AuNPs) have interesting physical and optical property on surface plasmon resonance (SPR). Especially, AuNPs display different absorption spectra when the AuNPs possess different sizes, shapes, or being functionalized with chemical molecules. In this study, an optical biosensing platform was established and was based on the SPR property of AuNPs for the measurement of proteinase activity.
First, the 13 nm AuNPs was modified with gelatin as proteinase substrate and then modified with 6-mercapto-1-hexanol (MCH) as an inducer. When AuNPs was modified with gelatin and MCH (AuNPs/MCH-gelatin), the gelatin increased the steric repulsion of AuNPs, and prevented the AuNPs surfaces from coming into close contact. For this reason, the AuNPs/MCH-gelatin could disperse in solution, and exhibited a dramatic stability in strict environment. After the proteinase (trypsin or matrix metalloproteinase-2 [MMP-2]) digested the substrate on AuNPs/MCH-gelatin, the AuNPs lost shelter and MCH increased the
attraction force between AuNPs. Therefore, the AuNPs were close to each other and
gradually resulted in aggregation. The AuNPs aggregation could be monitored by a red-shift in surface plasmon absorption and color would change from wine-red to violet-purple.
Furthermore, the color change can be observed by naked eyes, and the maximum wavelength (λmax) was measured by the UV/Vis spectroscopy.
In the AuNPs-based optical biosensing platform, the absorption ratio (A625 nm/A525 nm) of AuNPs was used to quantitively estimate the proteinase activity, and a linear range of trypsin detected by the AuNPs-based optical biosensing platform was from 5 × 10-1 to 5 × 102 U and with a detection limit of 5 × 10-1 U. A linear correlation was established when the MMP-2 activity was from 50 ng/mL to 600 ng/mL, and with a detection limit of 50 ng/mL. Besides, two MMP-2 inhibitors, galardin and ONO-4817, were used in this study for
feasibility analysis of drugs screening. In the AuNPs-based method, the MMPs activity for IC50 values of galardin and ONO-4817 were 1.87 nM and 17.76 nM, respectively. In comparision with zymography, MMP-2 activity for IC50 values of galardin and ONO-4817 were 3.48 and 14.33 nM, respectively. The result of the AuNPs-based method was consistent with that of zymography method. Furthermore, the AuNPs-based method can shorten detection time to less than 30 min. Thus, the rapid and sensitive novel AuNPs-based optical biosensing platform had potential for further applications in anti-MMPs drug
screening and for MMP-related diseases diagnosis.
Keywords: Gold nanoparticles, Proteinase, Optical biosensing platform, Matrix
誌 謝
兩年的碩士生涯，即將隨著此頁落幕，接著邁向人生新的里程碑。其實沒想過最後 的學生生涯會在交大渡過，從一開始考程的排定，選錯考科，到備取備上，一波多折的 過程，我想，是命中註定該到此地渡過這兩年的時光吧！ 首先感謝我的指導教授林志生老師收留我這個備取生，在我研究所期間以自身嚴謹 的態度來教導學生，在我學習過程中的諄諄教誨，使我在研究所期間獲益良多，在此獻 上最誠摯的感謝。此外也感謝顏聰榮教授、楊昀良教授、蘇平貴教授、溫曉薇教授及吳 啟華教授，謝謝各位老師撥冗來參加我的碩士學位口試，給予學生寶貴的建議，使得這 本論文能更臻完善。 感謝已有幸福家庭的建龍學長、好好先生俊旭學長、雖已畢業但還是很常見面的宜 貞學姐；帶領Biosensor 組獲得不少獎項的思豪學長、提供住所讓大家吃火鍋的棠青學 長、總是在電話中吵架的筱晶學姐、常被我問問題的聖壹學長、欲研發棗子芭的曜禎學 長；同為宅大三人組愛摸人一把的千雅同學、暫當助理的証皓同學、藻類扛霸子之一的 明達同學；碩一正妹群：庭妤、郡誼、子慧和瀞韓學妹；每次聚會出遊都會跟到的唯婷 學妹與修兆學弟；從SPCE 世界開始陪伴的欣儒學妹。感謝你們在這兩年間於實驗上及 生活中各事務的幫助，使我的研究生活變得絢麗多彩，謝謝你們。 感謝隔壁實驗室與我稱兄道弟的雯雯阿布兄，很妙的認識過程，謝謝妳於兩年中跟 我一起練瘋話，希望往後彼此一路順遂；好友佳穎、舞喵、乙禾老大，跟我一起環島的 水餃王振暉，衷心謝謝妳們包容愛亂跳tone 的我，並感謝你們曾在我低潮難過時陪伴我 渡過。 最後，僅以此論文獻給我最親愛的家人，我的母親跟我的阿姨，謝謝你們的支持， 使我得以完成我的碩士學位，願與你們分享這份榮耀！ 李榕均 謹誌 國立交通大學 生物科技學系碩士班 中華民國九十八年七月Content
Chinese abstract ... i
English abstract ...iii
Acknowledgment ... vi
Content ... vi
List of Tables ... vi
List of Figures ... vi
I. Literature Review ... 1
1-1 Biosensors... 1 1-1-1 Analytical devices... 1 1-1-2 Transducer ... 2 1-1-3 Bio-recognition element ... 2 1-1-4 Applications of biosensors... 3 1-1-5 Properties of biosensors... 3 1-2 Gold nanoparticles ... 41-2-1 Wet chemical synthesis techniques for small spherical particles ... 5
1-2-2 Synthesis of AuNPs by ultrasound ... 7
1-2-3 Synthesis process of AuNPs ... 9
1-3 Surface modification and bioconjugation of AuNPs ... 9
1-4 Surface plasmon resonance of AuNPs ... 10
1-5 Nanoparticles stability - DLVO theory ... 12
1-6 Applications of AuNPs... 13
1-6-1 Applications of AuNPs in biosensors ... 13
1-6-2 Applications of AuNPs in drug delivery system... 15
1-6-3 Applications of AuNPs in bioimaging... 16
1-7 Matrix metalloproteinases... 17
1-7-1 Gelatinase A (MMP-2) ... 18
1-8 Activation of MMPs... 19
1-9 Substrate zymography... 20
II. Research Strategy... 23
3-1 Reagents and solutions... 26
3-2 Preparation of the AuNPs/MCH-gelatin ... 26
3-2-1 Synthesis of 13 nm AuNPs ... 26
3-2-2 Modification of AuNPs/MCH-gelatin ... 27
3-3 Confirm size and morphology change of AuNPs/MCH-gelatin... 28
3-3-1 Dynamic light scattering... 28
3-3-2 Scanning electron micrographs ... 28
3-3-3 The electrophoresis analysis of modified-AuNPs ... 29
3-4 Investigation the stability of AuNPs/MCH-gelatin... 29
3-5 Assay of proteinase activity ... 30
3-5-1 Activation of MMP-2 ... 30
3-5-2 Proteinase activity assay by AuNPs-based optical biosensing platform ... 31
3-5-3 Proteinase activity assay by zymography... 31
3-6 Assay the efficiency of MMPs inhibitors ... 32
3-6-1 Assay the efficiency of MMPs inhibitors by AuNPs-based optical biosensing platform... 32
3-6-2 Assay the efficiency of MMPs inhibitors by zymograph ... 33
IV. Results and Discussion... 34
4-1 Synthesis of AuNPs and AuNPs/MCH-gelatin... 34
4-2 Identification of the size and morphology change of modified-AuNPs ... 35
4-3 The electrophoresis mobility of modified-AuNPs... 36
4-4 Effects of MCH on AuNPs-based optical biosensing platform ... 37
4-5 The stability of AuNPs/MCH-gelatin ... 38
4-6 Assay of proteinase activity ... 39
4-6-1 Assay of trypsin activity by AuNPs-based optical biosensing platform... 39
4-6-2 Assay of MMP-2 activity by AuNPs-based optical biosensing platform ... 39
4-6-3 Assay of MMP-2 activity by zymography... 40
4-7 Assay the efficiency of MMPs inhibitors ... 40
V. Conclusions ... 42
List of Tables
Table 1-1. Common transducers used in biosensing systems ... 59
Table 1-2. The applications of biosensors... 60
Table 1-3. The applications of AuNPs ... 62
Table 1-4. Prognostic value of mmps in cancer ... 63
List of Figures
Figure 1-1. The principle of biosensing systems... 67
Figure 1-2. Schematic presentation of the surface plasmon resonance... 68
Figure 1-3. UV-Vis absorption spectra of colloidal AuNPs with different diameters... 69
Figure 1-4. The DLVO theory... 70
Figure 2-1. A schematic illustration of the AuNPs-based optical biosensing Figure 2-1. platform to assay proteinase activity ... 71
Figure 2-2. The experimental flowchart of study strategies... 72
Figure 4-1. UV-Vis absorption spectra of 13 nm AuNPs and modified-AuNPs... 73
Figure 4-2. The size change of modified-AuNPs... 74
Figure 4-3. Investigating the morphology change of modified-AuNPs... 75
Figure 4-4. The electrophoresis mobility of modified-AuNPs ... 76
Figure 4-5. The effect of MCH on AuNPs-based optical biosensing platform... 77
Figure 4-6. Time-dependent changed of absorbance spectrum of AuNPs-based Figure 4-6. optical biosensing platform ... 78
Figure 4-7. The effect of different concentration of MCH on AuNPs-based optical Figure 4-7. biosensing platform ... 79
Figure 4-8. The stability of AuNPs/MCH-gelatin in strict environment... 80
Figure 4-9. Colorimetric assay for trypsin by using AuNPs/MCH-gelatin... 81
Figure 4-10. Absorption spectra of AuNPs/MCH-gelatin in the presence of trypsin ... 82
Figure 4-11. Absorption spectra of AuNPs/MCH-gelatin in the presence of MMP-2... 83
Figure 4-12. Detection of MMP-2 activity by zymography... 84
Figure 4-13. Screening inhibitors of MMP-2 by AuNPs-based optical biosensing Figure 4-14. platform ... 85
I. Literature Review
1-1 Biosensors
Since the glucose sensor was reported by Clark and Lyons in 1962 [Clark and Lyons, 1962], which generally recognized as the first biosensor, many types of biosensor and their associated techniques have been studied and developed [Bergveld, 1996; Nakamura and Karube, 2003]. According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), the biosensor is the device that uses specific biological components detect of an analytic with a physicochemical detector component. Biosensors are useful tools for investigation of bio-molecular interactions [Boozer et al., 2004; Campbell et al., 2004; Bollmann et al., 2005; Buchmueller et al., 2005]. The interaction between the analyte and the biological component is designed to produce a physicochemical signal that can be measured by the transducer and can be converted into a measurable effect such as an electrical signal [Vo-Dinh and Cullum, 2000]. Figure 1-1 illustrates the conceptual principle of the
biosensing process.
1-1-1 Analytical devices
The biosensor analytical devices combine a biological material (e.g., tissue,
microorganisms, organelles, cell, cell receptors, enzymes, antibodies, nucleic acids, etc.) [Cosnier et al., 2004; Davidson et al., 2004; Davis et al., 2005], intimately associated with or integrated within a physicochemical transducer which could be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical, and that is primarily responsible for the display of the results in a user-friendly way [Cavalcanti et al., 2008].
1-1-2 Transducer
The transducer and the detector element play an important role in the detection process. Depending upon the variety form of signal resulting from the interaction between the analyte and the biological element, the transducer can be classified into electrochemical, optical, piezoelectric, thermal, etc.(Table 1-1). Furthermore, novel types of transducers are constantly being developed for use in biosensors. The signal can be transformed into another signal that can be more easily measured and quantified. For a given
analyte-recognition element reaction, several transduction schemes may be applicable. However, constraints may be imposed by the intended use [Feriotto et al., 2004; Fojta et al., 2004; Fu et al., 2005]. A transducer should be not only highly specific for the analyte of interest, it should be able to respond in the appropriate concentration range and have a moderately rapid response time. The transducer also should be reliable, able to be
miniaturized, and suitably designed for practical application [Foulds and Lowe, 1985; Alocilja and Muhammad-Tahir, 2008].
1-1-3 Bio-recognition element
Biosensors consist of bio-recognition system, typically enzymes or binding proteins, such as antibodies, immobilized onto the surface of physico-chemical transducers.
Immuno-sensors are often used to describe biosensors which use antibodies as their
bio-recognition system [Graham et al., 2004; Gronewold et al., 2005; Halder et al., 2005].
In addition to enzymes and antibodies, the bio-recognition system can also include nucleic acid, bacteria, single cell organisms, and even whole tissues of higher organisms. Specific interactions between the target analyte and the complementary bio-recognition layer produce a physico-chemical change which is detected and may be measured by the transducer
[Knight, 2004; Krieg et al., 2004; Ladd et al., 2004]. In principle, any bio-molecule or molecular assembly that has the capability of recognizing the analyte can be used as a bio-receptor.
1-1-4 Applications of biosensors
Biosensors are used in increasingly broader ranges of application. The following describes some of the current applications, clinical diagnosis and biomedicine, farm and veterinary analysis, process control (fermentation control and analysis), food and drink production analysis, microbiology (bacterial and viral analysis), pharmaceutical and drug analysis, industrial effluent control, pollution control and monitoring, mining and toxic gases, and military applications. The applications of biosensors are listed in Table 1-2.
Biosensors are beginning to move from the proof-of-concept stage to field testing and commercialization in the United States, Europe, and Japan. Biosensors have several characteristic properties. The following section describes properties of biosensors [Cho et al., 2004; Li et al., 2005].
1-1-5 Properties of biosensors
Specificity: Like other bio-analytical methods (such as immuno-assays and enzyme assays),
biosensors use a biologically derived compound as the sensing element. The advantage of biological sensing elements is their remarkable ability to distinguish between the analyte of interest and similar substances. With biosensors, it is possible to measure specific analytes with great accuracy.
Speed: One characteristic of biosensors that distinguishes them from other bio-analytical
measured. There is no need to wait for results from lengthy procedures carried out in centralized laboratories.
Simplicity: The uniqueness of a biosensor is that the receptor and transducer are integrated
into one single sensor. This combination enables the measurement of target analytes without using reagents. For example, the glucose concentration in a blood sample can be measured directly by a biosensor (which is made specifically for glucose measurement) by simply dipping the sensor in the sample. This is in contrast to the conventional assay in which many steps are used and each step may require a reagent to treat the sample.
Continuous monitoring capability: Another advantage of biosensors is that the
bio-analytical assays can regenerate and reuse the immobilized biological recognition element. For enzyme-based biosensors, an immobilized enzyme can be used for repeated assays. This feature allows these devices to be used for continuous or multiple assays. By contrast, immunoassays, including enzyme-linked immunosorbent assay (ELISA), are typically based on irreversible binding and are thus used only once and discarded.
1-2 Gold nanoparticles
Nanoparticles, especially gold nanoparticles (AuNPs), have received great interests due to their attractive electronic, unique optical, thermal and physical properties as well as catalytic properties and potential applications in the fields of bio-nanotechnology (such as drug and gene delivery, and bioimaging) and in the rapidly developing area of biosensors [Park et al., 2002; Guo and Wang, 2007; Wang et al., 2008]. Therefore, the synthesis and characterization of AuNPs have attracted considerable attention. Furthermore, they have been proposed as future building blocks in nanotechnology [Persoons and Verbiest, 2006;
Zhao et al., 2008]. Table 1-3 shows the applicationof AuNPs.
Since the first synthesis report of AuNPs appeared about 150 years ago, numerous preparative methods leading to monodisperse particles of adjustable size and shape have surfaced. The common method for synthesis of AuNPs is wet chemical synthesis. Another method for the experimental generation of AuNPs is by sonolysis. The details of synthesis methods, characteristic, and application of AuNPs are as below:
1-2-1 Wet chemical synthesis techniques for small spherical particles
The common method for synthesis of AuNPs is wet chemical synthesis. In a typical synthesis of AuNPs, gold salts such as AuCl3 are reduced by the addition of a reducing agent which leads to the nucleation of Au ions to nanoparticles. Furthermore, a stabilizing agent is also required for stabilize the AuNPs. The size and shape of nanoparticles greatly influences their properties [Rao and Cheethama, 2001]. For example, spherical AuNPs exhibit a single plasmon resonance in the visible region of the spectrum, while rodlike particles exhibit a longitudinal and transversal plasmon resonance [Hutter and Fendler, 2002]. The common wet chemical synthesis methods of AuNPs including the citrate reduction method and the Brust method.
The citrate reduction method: The citrate reduction method was proposed by Turkevich in
1951 and this is the most well-known and simplest method for synthesizing gold colloids. This method is used to produce modestly monodisperse spherical AuNPs involving the reduction of HAuCl4 by sodium citrate in water. A typical standard citrate reduction procedure to fabricate AuNPs with an average diameter of 20 nm is as follows [Frens 1973; Grabor et al., 1997; Glomm, 2005; Persoons and Verbiest, 2006]:
boiled in reflux conditions under vigorous stirring and secondly 10 mL of 38.8 mM aqueous sodium citrate was quickly added to the HAuCl4 solution. This reaction resulted in color changes of the originally yellow solution to dark blue/grey. After 2 min, the color of solution became wine-red, indicating the end of the reaction. This mixture was further stirred and boiled for 15 min and subsequently cooled to room temperature while stirring continuously. The resulted colloidal AuNPs are approximately spherical and have an overall negative surface charge due to the citrate coverage. In the reaction, the citrate ions reduce the gold salt HAuCl4 according to
3 (H2CCOOH)2C(OH)COO− + 2AuCl−4 ↔ 3 (H2CCOOH)2C=O + 2Au + 8Cl− + 3CO2 + 3H+
The gold colloids are stabilized by negatively charged citrate ions and chloride ions that are still present in the solution. The citrate is not only as a reductant but also as a kinetic stabilizer. Irreversible aggregation or coagulation is easily induced by addition of
electrolytes (e.g. KI, NaCl, and KNO3) to the solution. The AuNPs size can be control by changing the concentration of the added sodium citrate [Frens, 1973]. To synthesis larger particles, less sodium citrate should be added. However, the results are less reproducible, the larger particles are less monodisperse and the color of the solution is violet, indicating the importance of the citrate ions stabilizing the gold colloids [Glomm, 2005; Persoons and Verbiest, 2006]. The AuNPs are stabilized by electrostatic repulsion due to adsorbed citrate ions on their surface that impart negative charge to the nanoparticles [Turkevich, 1985; Nath and Chilkoti, 2004].
The Brust method: This two-phase synthesis method was described by Brust and Schiffrin in
1994, and can be used to synthesize AuNPs in organic liquids that are normally not miscible with water [Brust et al., 1994].
having thiol, amide or acid groups in the solutions. The stabilization with organic molecules having thiols is due to the covalent bond that gold binds specifically to the sulfur atom of the thiol group [Rodriguez et al., 2003] while the organic molecules forms the actual stabilization preventing the particles to aggregate. The main advantage of the Brust method is that the AuNPs behaves like chemical compounds [Whyman, 1996]. The AuNPs can be precipitated, filtered off and redissolved in organic solutions. Furthermore, several stabilization agents with thiol, amide or acid groups can be used to sterically stabilize the gold colloids. The preparation processes is as follows [Brust et al., 1994]:
First, 30 mL of a 30 mM aqueous solution of HAuCl4 was mixed with a solution of tetraoctylammonium bromide (TOAB or TOABr) in 80 mL 50 mM toluene (C6H5CH3) and stirred vigorously. After the tetrachloroaurate was transferred into the organic layer, the l70 mg dodecanethiol was then added to the organic phase. Second, 25 mL of a freshly prepared 0.4 M aqueous solution of sodium borohydride (NaBH4) was slowly added with vigorous stirring. After further stirring for 3 hr the organic phase was separated, and evaporated to 10 mL in a rotary evaporator. To remove the excess of thiocholesterol, the organic phase was mixed with 400 mL ethanol. The mixture is then kept at -18°C for 4 hr and the dark brown precipitate was filtered off and washed with ethanol. The crude product was dissolved in 10 ml toluene and again precipitated with 400 ml ethanol. The overall reaction is as follows [Brust, 1994]:
AuCl4− (aq) + N(C8H17)4+ (toluene)
→
N(C8H17)4+ AuCl4− (toluene) mAuCl4− (toluene) + nC27H45SH (toluene) + 3me− →4mCl− (aq) + (Aum) (C27H45SH)n (toluene)
1-2-2 Synthesis of AuNPs by ultrasound
method can effectively form gold complexes and only after the addition of a suitable reducing agent to the sonicated solution will the formation of AuNPs be observed. The mechanism of the fabrication of the AuNPs depends on the pyrolysis of water and other organic compounds present in the aqueous solution resulting in the formation of free radicals at high temperatures and pressures. When water is sonicated in the presence of ethanol, the following reactions proceed [Okitsu et al., 2001; Caruso et al., 2002]:
H2O
→
•H + •OHCH3CH2OH + •H(•OH)
→
CH3CH•OH + H2 (H2O) CH3CH2OH→
pyrolysis radicalsThese radicals can reduce gold(III) ions into gold(II), gold(I), and finally gold(0). When the AuCl4− is sonicated in water without the addition of ethanol, some AuNPs is
produced according to three separate near diffusion-controlled one-electron transfer steps with H• as the primary reducing species [Caruso et al, 2002]:
AuCl−4 + 3H•
→
Au(0) + 4Cl− + 3H+And in the presence of ethanol a more complex sequence of three separate one-electron transfer reactions may be summarized [Okitsu et al, 2001; Caruso et al, 2002]:
3CH3CH•OH + AuCl4−
→
3CH3CHO + Au(0) + 4Cl− + 3H+The reduction of AuCl4− to colloidal gold according to the above two reactions is simplified and the particle growth is much more complex in the real sample solution. The rate of gold(III) reduction can be controlled by the ultrasound irradiation conditions such as the temperature and the ultrasound intensity. The size of the AuNPs can be controlled by
changing the alcohol concentration and alkyl chain length [Caruso et al, 2002]. This method is useful in the rapid fabrication of AuNPs, but the particles are polydisperse which is a problem for applications where monodisperse solutions are required [Persoons and Verbiest, 2006].
1-2-3 Synthesis process of AuNPs
The synthesis process of AuNPs involves three distinct stages: nucleation, growth and coagulation [Turkevich, 1985; Goia and Matijevic, 1998; Goia and Matijevic, 1999].
In the first stage, nucleation, metal ions are reduced to metal atoms, and rapid collisions to form stable icosahedral nuclei of 1-2 nm in size. The factors that affect the initial
concentration of nuclei include the following: the concentration of the reducing agent, the solvent, temperature and reduction potential of the reaction. Increasing the molar ratio of reducing agent to metal salt causes rapid formation of a large number of nuclei and leads to smaller, monodisperse AuNPs. In contrast, decreasing the molar ratio leads to slow formation of a few nuclei, and results in larger AuNPs with a greater heterogeneity in size. This stage is important for controling the shape, size and structure distribution of AuNPs and is typically complete in a few seconds. In the growth stage, the metal ions are reduced on the surface of the nuclei, until all the metal ions are consumed. The final stage for synthesis of AuNPs is coagulation, which involves prevention of AuNPs aggregation by the addition of stabilizing agents, which is either adsorbed or chemically bound to the surface of the AuNPs, and is typically charged. The equally charged AuNPs repel each other so that they are colloidally stable [Sperling et al., 2008].
1-3 Surface modification and bioconjugation of AuNPs
AuNPs are surrounded by a shell of stabilizing molecules. One end of these stabilizing molecules are either adsorbed or chemically linked to the gold surface, while the other end points towards the solution and provides colloidal stability [Sperling et al., 2008]. After synthesis of AuNPs, the stabilizer molecules can be replaced by other stabilizer molecules in a
ligand exchange reaction [Pellegrino et al., 2005].
Biological molecules can be attached to the AuNPs in several ways. If the biological molecules have a functional group which can bind to the gold surface (e.g., -SH, -CN, -NH2, -COOH, -OH), the biological molecules can replace some of the original stabilizer molecules when they are added directly to the AuNPs solution [Grabar et al., 1995; Kumar et al., 2004; Sperling et al., 2008]. By choosing the suitable molecules, it is possible to adjust the surface properties of the particles and attach different kinds of molecules to the AuNPs.
1-4 Surface plasmon resonance of AuNPs
AuNPs have emerged as important colorimetric reporters because of their high
extinction coefficients and strongly distance-dependent optical properties [Kim et al., 2001; Huang et al., 2005], the interesting optical and electronic properties that have served as a versatile platform for exploring many facets of basic science. AuNPs can strongly absorb and scatter visible light. When visible light shines on AuNPs, the light of a resonant
wavelength is absorbed by AuNPs and the visible light energy excites the free electrons in the AuNPs. The phenomenon induces surface electron oscillation of AuNPs and is responsible for the intense colors exhibited of AuNPs, the so-called surface plasmon resonance (SPR) [Sönnichsen et al., 2002; Nath and Chilkoti, 2004; Sperling et al., 2008; Zhao et al., 2008]. The schematic presentation of the SPR is shown in Figure 1-2. And the SPR depends
strongly on the size, shape, medium, and the relative distance of the AuNPs [Su et al., 2003; Sun and Xia, 2003; Schultz, 2003]. The Figure 1-3 shows UV-Vis absorption spectra of
AuNPs with different diameters.
absorption band (surface plasmon band) at about 520 nm in the visible light spectrum, resulting in the AuNPs display red in color. When the AuNPs are small, the surface electrons are oscillated by the incoming light in a dipole mode. As the size of AuNPs increases, the light can no longer polarize the nanoparticles homogeneously. Hence, the higher order modes at lower energy dominate. This causes a red-shift and broadening of the surface plasmon resonances. Therefore, the surface plasmon band red shifts with increasing AuNPs size [Ghosh and Pal, 2007; Zhao et al., 2008]. The red shift and color change also can be observed during the aggregation of small AuNPs. The spherical AuNPs with
interparticle distance higher than the average particle diameter appear red in color. When the interparticle distance become smaller than the average particle diameter, their surface
plasmons combine (interparticle plasmon coupling), and the aggregate could be considered as a single large particle, resulting in color changes from red to blue [Link and El-Sayed, 1999; Jena and Raj, 2008; Zhao et al., 2008]. The interparticle plasmon coupling can generate a huge absorption band shift (up to ~300 nm), and the color change can be observed by the naked eye. Therefore, complicated instruments are not required for analysis.
This unique optical property of AuNps can provide an elegant colorimetric platform for detection biological molecular interaction. When target analyte or a biological molecular which directly or indirectly triggers AuNPs aggregation (or redispersion of an aggregate), this process can be detected by the color change of the AuNP solution. The ratio of the
absorbances at 520 nm (for 13 nm AuNPs), which corresponds to dispersed particles, and a longer wavelength (e.g., 600 nm), which corresponds to aggregated particles, is often used to quantify the aggregation process or color change [Zhao et al., 2008].
1-5 Nanoparticles stability - DLVO theory
DLVO theory was developed by Derjaguin, Landau, Verwey and Overbeek in the 1940s, and has been used to explain the stability of colloids in suspension. The theory describes the force between charged surfaces interacting through a liquid medium. The stability of
colloidal system is determined by the balance between two opposing forces: electrostatic repulsion and van der Waals attraction [Craig et al., 1998; Malvern Instruments]. The particle interaction forces are described by Figure 1-4.
The dominant cause of aggregation is the van der Waals attractive forces between the particles, which are long-range forces [Shaw, 1980]. Van der Waals attraction is actually the result of attractive forces acting between individual particles in colloid system. The effect is accumulative. One particle of the first colloid has a van der Waals attraction to each particle in the second colloid. This phenomenon is repeated for each particle in the first colloid, and the sum of all of these is the total attractive forces. The variation in van der Waals force with distance between the particles is demonstrated by an attractive energy curve [Zeta-Meter, Inc. 1993; Pashley and Karaman, 2005].
The DLVO theory proposes that an energy barrier resulting from the repulsive force prevents particles aggregation. The particles will suspension in the solution, and the system will be stable. However, if the particles collide with sufficient energy to overcome that barrier, the attractive force will pull them approaching and adhering each other, the particles will be irreversibly aggregation. An electrostatic repulsion curve is used to indicate the energy that must be overcome if the particles are to be forced together. The maximum energy is related to the surface potential and the zeta potential. Therefore, the repulsive forces plays an important role in maintain the stability of the colloidal system. There are two essential mechanisms that affect dispersion stability.
Steric repulsion: The stability of colloidal dispersions can be enhanced by the addition of
suitable material (protective agents) adsorbing or otherwise attaching to the particle surfaces and preventing the particle surfaces coming into close contact [Shaw, 1980; Persoons and Verbiest, 2006]. If enough material adsorbs to the particles, the coating is sufficient to keep particles separated by steric repulsions between the polymer layers, and the van der Waals forces are too weak to cause the particles to aggregate. The mechanism of steric
stabilization is simple, requiring just the addition of a suitable material. However, the material can be expensive and in some cases the material is undesirable for subsequently aggregate the system if that is required [Malvern Instruments].
Electrostatic or charge stabilization: The effect on particle interaction results from the
distribution of charged species in the colloid system. Energy is required to overcome this repulsion. An electrostatic repulsion curve is used to indicate the energy that must be overcome if the particles are to be forced together. The maximum energy is related to the surface potential and the zeta potential. This mechanism can stabilize or aggregate the particles in the system by altering the concentration of ions in the surrounding. This process is simple and potentially inexpensive [Zeta-Meter, Inc. 1993; Pashley and Karaman, 2005; Malvern Instruments].
1-6 Applications of AuNPs
1-6-1 Applications of AuNPs in biosensors
AuNPs have great potential applications in the field of biosensors due to their display many interesting electrical and optical properties. AuNPs can exhibit surface plasmon resonance and plasmon absorption in the red-shift due to interparticle plasmon interactions
[Wang et al., 2009]. This optical property of AuNPs can establish a highly selective and sensitive colorimetric assays for molecular recognition events [Rosi and Mirkin, 2005; Jena and Raj, 2008]. AuNPs-based colorimetric assays for detection are mainly dependent on the analyte induce the change of interparticle distance of AuNPs, and resulting in color change of AuNPs. Since the first AuNPs-based DNA sensor was developed by Mirkin and coworkers [Mirkin et al., 1996], the AuNPs-based platform has been increasingly applied for the
detection of a large variety of targets, including nucleic acids [Cao et al., 2005; Li et al., 2005; Chen et al., 2008], proteins [Tsai et al., 2005; Chen et al., 2008], enzyme activity [Xu et al., 2007; Jiang et al., 2009] and metal ions [Liu and Lu, 2007; Slocik et al., 2008].
Besides, the exceptional quenching ability of AuNPs makes them excellent materials for Förster resonance energy transfer (fluorescence resonance energy transfer; FRET)-based biosensors [Sapsford et al., 2006; De et al., 2008]. The fluorescence of many fluorophores is quenched when they are in close proximity to AuNPs surface [Dulkeith et al., 2002; Sperling et al., 2008]. For detection of analyte, AuNPs are conjugated with ligands that specifically bind to the analyte, and analyte molecules are modified with fluorophores. When the analyte and ligand have interaction, the fluorophores are closely linked to the AuNPs and their
fluorescence is quenched. Another detection scheme works slightly differently. In this case a molecule is used as a spacer to link fluorophores to AuNPs. In the presence of the analyte, the spacer molecule changes its conformation, resulting in quenching or releases the fluorescence of the fluorophore [Sperling et al., 2008]. The FRET-based biosensors has been used for the detection of nucleic acids [Maxwell et al., 2002; Ray et al., 2006] and proteins [Oh et al., 2005; Oh et al., 2006; Huang et al.,2007].
Finally, AuNPs can also be used for the transfer of electrons in redox reactions, due to their conductivity and catalytic property [De et al., 2008; Sperling et al., 2008]. In the electrochemical biosensor, enzyme can specifically oxidize (or reduce) the analyte molecules,
and the flow of electrons released (or required) in this redox reaction can be measured as electrical current. Therefore, enzyme is conjugated with the AuNPs [Xiao et al., 2003], and immobilized on the surface of an electrode which is connected to an amplifier for current detection. Alternatively, AuNPs also can be first immobilized on the electrode and then modified with enzymes [Xiao et al., 1999]. The electrode covered with a layer of AuNPs has a larger surface area, and the enzyme conjugated with the AuNPs can facilitate the electron transport, these lead to enhance the currents [Sperling et al., 2008].
1-6-2 Applications of AuNPs in drug delivery system
AuNPs have been used for a long time for drug delivery systems (DDSs). Biology molecules are adsorbed on the surface of AuNPs and introduced into the cells. The
methods for particles introduction into a cell can either be forced as in the case of gene guns or achieved naturally by particles ingestion. These molecules will eventually detach themselves from the surface of the AuNPs when inside the cells [Sperling et al., 2008].
The method of gene guns is using AuNPs as massive nanobullets for ballistic projectile introduction of DNA into cells. DNA is adsorbed onto the surface of AuNPs which are then shot into the cells. This method has been used successfully for gene delivery [Tischer et al., 2002; Lee et al., 2008].
Another method for AuNPs application of drug delivery is achieved naturally by particles ingestion. As good biocompatible materials, AuNPs can be modified with
bio-molecular and would not destroy their biological activity. Furthermore, AuNPs can be uptaken by cells naturally, either specifically (via receptor-ligand interaction) or
nonspecifically [Chithrani et al., 2006; Sperling et al., 2008]. For specific uptake, the
ligands specific to receptors on the cell membrane are conjugated to the surface of the AuNPs. In this way, ligand-modified AuNPs are predominantly incorporated by cells which possess
receptors for these ligands, and is more effective than nonspecific uptake [Sperling et al., 2008]. It is possible to direct particles specifically to cancer cells by conjugating them with biomarkers on the surface of cancer cells but that are less present on healthy cells [Jain et al., 2007]. After ingestion, the AuNPs are stored in vesicular compartments inside the cells [Chithrani and Chan, 2007]. In order to release the particles from the vesicular structures to the cytosol, their surface can be coated with membrane-disruptive peptides or relying on the acidic condition inside tumor and inflamed tissues (pH~ 6.8) and cellular compartments including endosomes (pH~ 5.5-6) and lysosomes (pH~ 4.5-5.0) [de la Fuente and Berry, 2005; Yang et al., 2005]. AuNPs uptake-mediated delivery of molecules into cells is used mainly for two applications. First, gene therapy DNA is introduced into cells, which subsequently causes the expression or down-expression of the corresponding proteins [Sullivan et al., 2003; Salem et al., 2003]. Second, targeting anti-cancer drugs are specifically delivered to cancer tissue [Rojo et al., 2004; Jain et al., 2007].
1-6-3 Applications of AuNPs in bioimaging
AuNPs have been primarily used for bioimaging, based on the interaction between AuNPs and light. AuNPs are a very attractive contrast agent as they can be visualized with a large variety of different techniques [Sperling et al., 2008].
Immunostaining is one of the traditional methods that uses AuNPs in biology. Firstly, AuNPs are conjugated with antibody for molecular recognition. The antibody-modified AuNPs will bind to the antigen or target regions containing the antigen. The
antibody-modified AuNPs are added to fixed and permeabilized cells. Targets outside as well as inside cells can be labelled with AuNPs in this way. The AuNPs then provide excellent contrast for TEM imaging with high lateral resolution and larger structures can also be imaged with optical microscopy [Faulk and Taylor, 1971; De Mey et al., 1982].
AuNPs are not only used for visualizing structures within single cells, but also applied for providing contrast in vivo to whole organs in animals and potentially in humans. Firstly, the AuNPs are conjugated with antibodies or ligands which bind as specifically as possible to the organ of interest in the animal. When modified AuNPs are injected into the bloodstream of animals, these particles label at the target organs via receptor-ligand interaction [Sperling et al., 2008]. AuNPs bound to the organ provide contrast for imaging and resolving the
structure of the organ. X-rays, which can penetrate skin and organs deep inside the body can be imaged or addressed for therapy [Hainfeld et al., 2004]. Furthermore, AuNPs have the predominance of causing less cytotoxic damage than quantum dots, and that makes AuNPs possess more potential to be applied in medical applications.
1-7 Matrix metalloproteinases
Matrix metalloproteinases (MMPs) are a family of extracellular zinc-dependent
endopeptidases [Birkedal-Hansen et al., 1993] that are able to degrade all components of the extracellular matrix (ECM), including fibrillar and non-fibrillar collagens, gelatin, fibronectin, laminin, and basement membrane glycoproteins [Fedarko et al., 2004; Lombard et al., 2005]. MMPs not only play an important role in ECM remodeling in physiologic situations, such as embryonic development, cell migration, tissue regeneration, wound repair, apoptosis,
angiogenesis, and inflammatory, but also in pathological conditions, including rheumatioid arthritis, osteoarthritis, atherosclerotic plaque rupture, tissue ulceration, and in involved in the processes of tumors metastasis and growth [Miyazaki et al., 1990; Koivunen et al., 1999; Bergers et al., 2000; Roeb and Matern, 2001; Jones et al., 2003]. The levels of MMPs can be determined in patient serum or urine, where levels elevated over a particular threshold can sometimes predict progression or prognosis (Table 1-4).
MMPs are generally divided into six groups, collagenases (MMP-1, -8, and MMP-13), stromelysins (MMP-3, -10 and MMP-11), matrilysins (MMP-7 and MMP-26), gelatinases (MMP-2 and MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) (MMP-14, -15, -16, -17, -24 and MMP-25) and others [Jones et al., 2003]. Classification and nomenclature of all the types of MMPs were listed in Table 1-5. Although MMPs are
subclassified based on their ability to degrade various proteins of the ECM, they also play other important roles such as the activation of cell surface receptors and chemokines
[Stefanidakis and Koivunen, 2006]. In addition, MMP-2 has proteolytic activity to specific targets within the cell to cause acute, reversible contractile dysfunction in cardiac disease [Schulz, 2007].
The regulation of MMPs occurs at many levels, including transcription (the major one), post- transcriptional modulation of mRNA stability, secretion, localization, zymogen
(proenzyme) activation and inhibition of activity by natural inhibitors of MMPs, tissue inhibitor of metalloproteinases (TIMPs). The TIMP gene family consists of 4 members: TIMP-1, -2, -3 and -4. TIMPs inhibit the activity of MMPs by binding to activated MMPs in a 1:1 molar stoichiometry [Brew et al, 2000]. TIMPs can also inhibit the growth, invasion and metastasis of malignant tumours [Vihinen and Kähäri, 2002].
1-7-1 Gelatinase A (MMP-2)
In 1978, Sellers et al. were first to separate a gelatinase activity from collagenase and stromelysin in the culture medium from rabbit bone [Seller et al., 1978]. Similar enzyme acting on basement membrane type IV collagen was reported by Liotta et al. [Liotta et al., 1979] in the following year. Gelatinase was purified from human skin, mouse tumor cells, rabbit bone, and human gingival. Gelatinase A was a triple repeat of fibronectin type I domains inserted in the catalytic domain; this domain participates in binding to the gelatin
substrates of the enzyme [Libson et al., 1995; Lee et al., 1997]. MMP-2 is ubiquitously expressed in the cells which comprise the heart and is found in normal cardiomyocytes, as well as in endothelium, vascular smooth muscle cells and fibroblasts [Coker et al., 1999]. In particular, MMP-2 is overexpressed in many cancers, including breast cancers, and is an indicator of cancer invasiveness, metastasis, angiogenesis, and treatment efficacy [Ratnikov et al., 2002].
1-8 Activation of MMPs
The MMPs are produced as zymogens. The basic structure of MMPs can be divided into three structural well-preserved domain motifs, including a catalytic domain, an
N-terminal domain and C-terminal domain. Zinc-dependent catalytic domain (about 170 amino acids) of MMPs contains a zinc binding motif HEXXHXXGXXH as the zinc binding active site, and having an additional structural zinc ion and 2-3 calcium ions, which are required for the stability and the expression of enzymic activity [Nagase and Woessner,1999; Jones et al., 2003]. The N-terminal domain (propeptide domain; about 80 amino acids) contains a unique PRCG(V/N)PD sequence in which the cysteine residue interacts with the catalytic zinc atom in the active site, prohibiting activity of the MMPs. Thus, the interaction has to be disrupted to “open” the cysteine switch in the process of MMPs activation [Van Wart and Birkedal-Hansen, 1990], which is a critical step that leads to ECM breakdown [Carmli et al., 2004]. The C-terminal hemopexin domain (about 210 amino acids) of MMPs has a four-bladed propeller structures and contributes to substrate specificity [Wallon and Overall, 1997]. In membrane-type MMPs, the hemopexin domain contains a
transmembrane domain for anchoring the protein in the membrane. Besides, the hemopexin domain in MMP-2 also has a function in the activation of the enzyme [Morgunova et al.,
1999].
All MMPs are synthesized in the latent form and require extracellular activation. MMPs can be activated in vitro by a variety of mechanisms. Activation of latent MMPs is believed to occur by dissociation of the sulfhydryl group of the cysteine from the active zinc site and replacement with a water molecule that plays a role in catalysis [Van Wart and
Birkedal-Hansen, 1990; Galazka et al., 1996]. Disruption of the Cys-zinc bond can be achieved by heavy metal ions, oxidants, organomercurials, sulfhydryl alkylating agents, or disulfide compounds [Murphy et al., 1980; Macartney and Tschesche, 1983; Weiss er al., 1985; Stricklin et al., 1983; Mallya and Van Wart., 1989]. Latent MMPs can also be activated by conformational changes of the polypeptide chain induced by detergents or chaotropic agents [Birkedal-Hansen and Taylor, 1982; Springman et al., 1990], and also by limited cleavage of the propeptide by proteolytic enzymes such as trypsin or chymotrypsin [Murphy et al., 1980; Stricklin et al., 1983; Okada et al., 1990].
1-9 Substrate zymography
Zymography and reverse zymography are described as sensitive, quantifiable, and functional assay to detect MMPs and TIMPs in biological samples [Leber and Balkwill, 1997; Hawkes et al., 2001]. All types of substrate zymography derive from gelatin zymography. The techniques are the same except that the substrate differs depending on the type of MMPs or TIMPs to be detected. The standard method is based on sodium dodecyl sulfate (SDS) polyacrylamide gels impregnated with a protein substrate, and proteins are separated by electrophoresis under denaturing conditions [Leber and Balkwill, 1997; Snoek-van Beurden and Von den Hoff, 2005].
After electrophoresis, the gel is washed, and exchange of the SDS with TritonX-100, which cause the enzymes partially renature and recover their activity. Additionally, the latent MMPs are autoactivated without cleavage. During incubation, the renatured MMPs in the gel will digest the substrate. After incubation, the gel is stained with CoomassieBlue, and the MMPs are detected as clear bands against a dark blue background of undegraded substrate. The clear bands in the gel can be quantified by densitometry [Heussen and Dowdle, 1980; Woessner, 1995; Hawkes et al., 2001]. Zymography is based on the following principles: First, during electrophoresis, gelatin is retained in the gel. Second, MMPs activity can be reversibly inhibited by SDS during electrophoresis. Finally, the SDS causes the separation of MMP-TIMP complexes during electrophoresis. This enables the detection of MMPs and TIMPs independently of one another [Leber and Balkwill, 1997; Hawkes et al., 2001].
A particular advantage of zymography is that both the proenzymes and the active forms of MMPs can be distinguished on the basis of their molecular weight. The inactive
proenzyme is about 10 kDa larger than the activated form. An additional advantage of zymography is that during electrophoresis the TIMPs dissociate from the MMPs, and do not interfere with detection of the enzymatic activity, which is not possible in solution assays [Woessner JF Jr. 1995; Leber and Balkwill, 1997]. However, there are many problems with the zymography. First, the limited number of wells per gel does not allow a full standard curve and several samples to be run on the same gel. Second, the refolding of MMPs after electrophoresis recovers only part of the original activity. Third, the two-step
staining/destaining method is not reliable and is difficult to reproduce. Traditionally, the destaining step is undefined length, usually several hours until a satisfactory background/band staining is achieved. Furthermore, overstaining of the gels reduces the assay sensitivity, because bands of low activity will become undetectable. In addition, excess destaining can
also bleach the bands, so that the intensity of gels cannot be assessed in the linear range of the assay [Leber and Balkwill, 1997; Snoek-van Beurden and Von den Hoff, 2005].
II. Research Strategy
Traditional methods for analyzing the activity of MMPs include the zymography and fluorescein-labeled synthetic peptides [Leber and Balkwill, 1997; Netzel-Arnett et al., 1991]. The zymography is time consuming and complicated for MMPs activity and inhibition studies. Moreover, fluorescein-labeled synthetic peptides increase the cost of diagnosis and need
expensive instruments to operate and analyze. Therefore, the SPR property of AuNPs is used to establish an AuNPs-based optical biosensing platform for measuring proteinase activity and screening the inhibitors of proteinase.
Most of AuNPs-based diagnoses for the detection of enzyme activity mainly depend on the enzyme properties to induce the change in AuNPs aggregation [Wang et al., 2006; Xu et al., 2007]. However, using AuNPs to establish a platform for detection of enzyme activity may encounter the following problems. First, ion concentration effects enzyme activity and AuNPs aggregation. When enzymes carry out their function, changing in the niche (such as pH, temperature, metal ion, or salt concentration) would affect the activity of enzymes;
therefore the diagnostic system should provide an adequate environment for enzyme working. In terms of AuNPs, AuNPs would easily aggregate while surface charge is neutralized by counterions. In previous AuNPs-based sensing systems, AuNPs aggregation relies on electrostatic interaction, while the surface charges of receptor-modified AuNPs become neutral upon the addition of target analyte [Sato et al., 2003; Zhao et al., 2007; Chen et al., 2008]. Combining above problems, when the AuNPs-based sening system is applied to assay enzyme activity, not only the analyte would promote monodispersed AuNPs to
aggregate, but also the cation which are supplied by the enzyme buffer may induce AuNPs to aggregate, it would result in false-positive results and interfere the analysis of enzyme
Moreover, according to the DLVO theory, the stability of colloidal system is determined by the balance between two opposing forces - electrostatic repulsion and van der Waals attraction [Craig et al., 1998; Malvern Instruments]. When the bio-recognition element was modified onto AuNPs as the substrate, the bioelement molecule provides the steric repulsion and prevents the AuNPs coming into close contact [Glomm, 2005; Persoons and Verbiest, 2006]. In a detection of protein-protein interaction like proteinase digestion, the steric hindrance between the proteins would lead AuNPs dispersion and the slow enzyme kinetics would prolong the detection time. In addition, AuNPs have high affinity for biomolecules [Lu et al., 2007], which can conjugate with amino acids that have thiol, amino, carboxylic, or hydroxyl groups in their side chains. Therefore, the protein which was digested by
proteinase would adsorb on AuNPs again and interfere with the aggregation of AuNPs.
To overcome these arduous problems, in this study, the colloidal AuNPs was modified with gelatin as proteinase substrate, and then modified with 6-mercapto-1-hexanol (MCH) not only as inducer but also for blocking of this system. Figure 2-1 illustrates the schema of this
platform and Figure 2-2 shows the experimental flowchart of this study.
When the AuNPs was modified with gelatin, the gelatin adsorbed onto the AuNPs surface not only as proteinase substrate but also increase the steric repulsion of AuNPs which can prevent the particle surfaces coming into close contact. After proteinase digested the AuNPs/gelatin, the AuNPs/gelatin still can suspend stably in the solution and retain red-wine due to the steric repulsion generated by gelatin was too difficult to overcome. Hence, the MCH) was used in this system for solve the problem.
The chemical compound, MCH, played an important role as an inducer. The MCH has two functional groups, one side is thiol group (-SH), and another is hydroxyl group (-OH). Both functional group of MCH can conjugate with AuNPs by covalent bond and the -SH group has stronger attraction with AuNPs than -OH group. Therefore, in the modified
process, the MCH predominantly functionalized with gelatin AuNPs by SH-Au covalent bond and the -OH group was been exposed on AuNPs surface [Sato et al., 2003; Chen et al., 2008]. In addition, the MCH can remove nonspecifically adsorbed of gelatin from the AuNPs surface, which helps to improve subsequent biomolecular recognition efficiency [Zhao et al., 2008].
When the AuNPs was modified with gelatin and MCH, the AuNPs/MCH-gelatin could suspend stably. After proteinase digested gelatin, the AuNPs/MCH-gelatin lost the shelter, the repulsion of steric hindrance generation by gelatin was decreased and MCH attracted other AuNPs by OH-Au covalent bond [Zhu et al., 2008]. MCH induced the AuNPs to
irreversibly aggregated and resulted in shift in the surface plasmon spectrum and a consequent color change of the AuNPs from red to purple. Besides, MCH modified on the AuNPs also plays the role as a blocker [Huang et al., 2007]. The MCH possessed the ability to bind on the surface of AuNPs where not conjugated with gelatin through the covalent bond, and prevent the gelatin which digested by proteinase to conjugate with AuNPs again.
In the system, the color change of AuNPs can be observed with the naked eye, and the maximum wavelength (λmax) can be measured by UV/Vis spectroscopy. In addition, the method could serve as an alternative platform for efficient screening of the proteinase
inhibitors. When the proteinase was inhibited by candidate drugs, the drugs block activity of proteinase, the AuNPs/MCH-gelatin are intact and stably in the solution without the color change. Therefore, the novel AuNPs-based optical biosensing platform can not only detect the activity of proteinase rapidly, but also can screen a great deal of effective inhibitor for proteinase.
III. Materials and Methods
3-1 Reagents and solutions
All chemicals were of analytical grade and were used without further purification. Sodium citrate (C6H5Na3O7·2H2O) was obtained from Merck (Darmstadt, Germany). Tris HCl was purchased from Chemicon (Invitrogen, San Diego, LA, USA). Sodium chloride (NaCl) was purchased from USB (Cleveland, OH, USA). Agaroseand 10 X
Tris-Borate-EDTA buffer (TBE) were purchased from Amresco (Cleveland, OH, USA). ONO-4817 (C22H28N2O6) was purchased from Tocris (Ellisville, MO, USA). Galardin (Ilomastat; GM6001; C20H28N4O4) was purchased from USBiological (Swampscott, MA). 40% acrylamide/Bis solution (37.5:1) was purchased from Bio-Rad (Hercules, CA, USA).
Calcium chloride (CaCl2), trypsin, 6-mercapto-1-hexanol (MCH), triton X-100, dimethyl sulfoxide (DMSO), p-Aminophenylmercuric Acetate (APMA), type A gelatin, glycine, hydrogen tetrachloroaurate(III) (HAuCl4 · 3H2O), coomassie brilliant blue R250, matrix
metalloproteinase-2 human (expressed in mouse NSO cells) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Nanopure water was obtained by passing twice-distilled water through a Milli-Q system (18 MΩ· cm; Millipore, Bedford, MA, USA).
3-2 Preparation of the AuNPs/MCH-gelatin
3-2-1 Synthesis of 13 nm AuNPs
literature procedure [Saraiva and de Oliveira, 2002]. A 50 mL aqueous solution consisting of 2.5 mM HAuCl4．3H2O was brought to a vigorous boil with stirring in a conical flask, and then 38.8 mM sodium citrate (5 mL) was added to the solution rapidly. This solution was boiled with vigorous stirring for another 15 min, resulted in the color change from the originally yellow solution to deep red. The solution was cooled to room temperature with continuous stirring for 15 min. The colloidal AuNPs with an average diameter of 13 nm was produced, and was stored at 4ºC.
Diameter of the prepared 13 nm AuNPs was measured by a scanning electron
microscope (SEM) and dynamic light scattering (DLS) particle size analyzer. Moreover, the absorption spectrum of AuNPs was measured by a spectrophotometer (SpectraMax 190; Molecular Devices Corporation, Sunnydale, CA, USA).
3-2-2 Modification of AuNPs/MCH-gelatin
The process of modified gelatin and MCH on AuNPs were monitored by observing the spectral change after the addition of gelatin and MCH to colloidal AuNPs. The gelatin and MCH were attached to the AuNPs according to below procedures:
The 50 μL of aqueous gelatin solution (0.1%) was added to 950 μL of the aqueous 13 nm AuNPs solution. After careful mixing, the mixture was incubated and shaken at 37°C for 2 hr. The mixture was then centrifuged for 6 min at 14,000 × g to remove the excess gelatin. After two centrifuge/wash cycles, the colloids was resuspended in 200 μL of NTTC buffer (50 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl2, and 0.05 % Triton X-100, pH 7.5), and this colloid solution was regarded as AuNPs/gelatin. The synthesis procedure of AuNPs/MCH-gelatin was similar with AuNPs/gelatin. After 950 μL AuNPs solution and gelatin (0.1%, 50 μL) incubated at 37°C for 2 hr, 10 μL of MCH (1 mM) was added into the solution. After
vortexing, the mixture was incubated and shaken at 37°C for another 2 hr. The mixture was then centrifuged for 6 min at 14,000 × g to remove the excess gelatin and MCH. After two centrifuge/wash cycles, the colloid solution was resuspended in 200 μL of NTTC buffer, and this colloid was regarded as AuNPs/MCH-gelatin.
3-3 Confirm size and morphology change of AuNPs/MCH-gelatin
3-3-1 Dynamic light scattering
Dynamic light scattering (DLS; also known as Photon Correlation Spectroscopy or Quasi-Elastic Light Scattering) is a technique which can be used to determine the size
distribution of small particles in solution. The method utilizes laser as light source which is monochromatic and coherent, and observes a time-dependent fluctuation in the scattering light intensity to determine the translational diffusion coefficient of small particles.
The AuNPs samples were diluted with NTTC buffer (filtered through 0.45 μm syringe filters) and filled into the light scattering cuvette. Light scattering experiments were
performed using the BI-200SM Goniometer (Brookhaven Instruments Corporation, Holtsville, NY, USA) at a temperature of 20°C. The laser wavelength was 532 nm, and measurements were conducted at an angle of 90°. The DLS data were analyzed by Brookhaven
Instruments-Dynamic Light Scattering software.
3-3-2 Scanning electron micrographs
High resolution scanning electron microscopic (SEM) images of modified-AuNPs were obtained with a field-emission SEM instrument JSM-6700F (JEOL, Tokyo, Japan) and operated at 15 kV. The samples were prepared by dropping 10 μl of AuNPs solution onto a
gold chip and incubated samples at 37°C for 30 min. Finally, the chips were rinsed thoroughly with distilled water and air-dried for scanning.
3-3-3 The electrophoresis analysis of modified-AuNPs
To observe the size change of modified-AuNPs which digested by proteinase, the gel electrophoresis was used to separate AuNPs according to the gelatin of attached.
Agarose gel 0.5 % (w/v) was prepared with and immersed in 0.5 X TBE buffer
(Tris-Borate-EDTA buffer, prepared by diluting 10 x stock solutions). The gel was run in a horizontal electrophoresis system (Mini-Sub Cell GT, Biorad, electrode spacing 15 cm). The gel image was recorded by a consumer digital camera and processed the images only with small linear contrast adjustments in order to give a true representation of the visual gel appearance.
Before loading the gel of the modified-AuNPs samples, the modified-AuNPs were coated with a layer of sodium dodecyl sulfate (SDS) which imparts a negative charge on the gelatin of AuNPs, and forced all of modified-AuNPs run to positive electrode. After trypsin digested at 37°C for 10 min, each of digested-AuNPs samples was loaded into one well of the gel, and the gel was ran for 30 min at 110 V in 0.5 X TBE buffer.
3-4 Investigation the stability of AuNPs/MCH-gelatin
To study the stability of AuNPs/MCH-gelatin in strict envionmrnt, 50 μL of different buffer solution was added into 200 μL of AuNPs/MCH-gelatin solution. The 1N HCl and the 1N NaOH were used to analyze the stability of AuNPs/MCH-gelatin under extreme pH
condition, and 10 X PBS buffer (1,370 mM NaCl, 27 mM KCl, 100 mM Na2HPO4, 20 mM KH2PO4, pH 7.4) was used to analyze the influence of high salt concentration buffer on AuNPs/MCH-gelatin. As a control, 50 μl of NTTC buffer solution was mixed with 200 μL of AuNPs/MCH-gelatin. All of samples were incubated at 37°C for 30 min, and the
absorbance wavelength was analyzed with UV-Vis absorption spectrophotometer.
3-5 Assay of proteinase activity
3-5-1 Activation of MMP-2
Preliminary experiments were undertaken to determine the concentrations MMP-2 required for maximal activation of each sample. The lyophilized powder MMP-2 was resuspended in 0.1 mL of TCNB buffer (composed of 50 mM Tris, 10 mM CaCl2, 150 mM NaCl2, and 0.05% triton-X100, pH 7.5), and activated by p-Aminophenylmercuric acetate (APMA).
APMA is an organomercurial agent used for the activation of latent MMPs in vitro. The procedure for activation was as follows: The stock solution of APMA was prepared by dissolve 3.5 mg APMA in 1 mL 0.1 M NaOH, this stock solution should be 10 mM. Then neutralize the high base by diluting 4 folds in Tris-Triton-Calcium buffer (50 mM Tris-HCl, 1 mM CaCl2, 0.05% triton X-100 pH 7.5). For activated MMP-2, the APMA solution was mix with MMP-2 sample to give a final concentration of 0.25 mM. Then activation times will vary depending upon the samples. MMP-2 generally requires short activation time. In this study, MMP-2 samples were activated at 37°C for 2 hr. This final activated MMP-2 solution can be used directly without dialyzing away the APMA [Sellers et al., 1977].
3-5-2 Proteinase activity assay by AuNPs-based optical biosensing platform
For the assay of proteinase activity, the measurement were performed in NTTC buffer as a control experiment and observed result. NTTC buffer would not influence the absorbance of AuNPs, and NTTC buffer would not promote the AuNPs/MCH-gelatin to aggregate. The concentration of gelatin modified-AuNPs was adjusted to 5 nM for the further assay of proteinase activity. In the proteinase activity assay, an amount of 50 μL trypsin or MMP-2 samples solution with different concentrations was added into 200 μL of modified-AuNPs and then the mixture was incubated at 37°C. The activity of MMP-2 were much lower than the ones of trypsin, in order to amplify the detection signal of MMP-2, the incubation time was adjusted to 30 min. After proteinase digested, 200 μL of mixture solutions were transferred into 96-well plate. All of samples were analyzed with UV-Vis absorption spectrophotometer and recorded their wavelength and A625 nm/A525 nm. The tests were performed in triplates.
3-5-3 Proteinase activity assay by
zymography
The activated MMP-2 solution was mixed with zymography buffer (composed of 0.5 M Tris-HCl, pH 6.8, glycerol, 10% (w/v) SDS, and 0.1% bromophenol blue) and stood at room temperature for 10 min. Then loaded on 8% SDS-polyacrylamide gel containing 0.1 mg/mL gelatin, and ran the gel with Tris-Glycine SDS running buffer at 80 V for 4 hr.
Following electrophoresis, the gel was washed twice in renaturing buffer (2.5% trixton X-100) with gentle agitation at room temperature for 30 min in order to exchange SDS to Triton-X100. And decant the renaturing buffer, then replaced with developing buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 5 mM CaCl2) at room temperature for 30 min, then replaced with fresh developing buffer and incubated at 37°C for 18 hr.
