A biocompatible synthesis of gold nanoparticles by Tris(hydroxymethyl)aminomethane
© Chen et al.; licensee Springer. 2014
Received: 25 November 2013
Accepted: 16 April 2014
Published: 7 May 2014
Gold nanoparticles' novel properties are widely realized in catalysis, plasmonics, electronics, and biomedical applications. For biomedical application, one challenge is to find a non-toxic chemical and/or physical method of functionalizing gold nanoparticles with biomolecular compounds that can promote efficient binding, clearance, and biocompatibility and to assess their safety to other biological systems and their long-term effects on human health and reproduction. In the present study, we describe a new method by using Tris(hydroxymethyl)aminomethane (Tris), a widely used buffer solvent of nucleic acid and proteins, as the reducing agent for synthesizing gold nanoparticles by one step. It is found that Tris carries out the reduction reactions in relatively mild conditions for biomacromolecules. Particularly, it can be used to modify the DNA during the process of preparation of gold nanoparticles. The morphology and size distribution of gold nanoparticles are consistent and were confirmed by many different approaches including dynamic light scattering (DLS), UV-visible (UV-vis) spectrophotometry, atomic force microscopy (AFM), and transmission electron microscopy (TEM).
Chemical and physical properties of gold nanoparticles are dependent of their sizes, shapes, and crystallinity . Up to now, most of the protocols for preparing gold nanoparticles (AuNPs) focus on the particles whose diameters range from 2 to 200 nm with various morphologies [2–5]. Due to their electrochemical properties, such as high affinity with biomolecules and their well-known optical absorption in the visible region surface plasmon band (SPB), gold nanoparticles are proved to be ideal nano-objects for medical imaging and even for photo-thermal therapy [6–8]. Therefore, the application of AuNPs in the biomedical field is growing exponentially. Despite a variety of reductants have been used to stabilize and synthesize AuNPs, only three approaches have been explored to produce size-defined gold nanoparticles through chemical reduction for medicinal applications. They are the citrate capping method , the biphasic Schiffrin-Brust method, and the seeding growth method [10, 11]. Reducing agents or stabilizers and synthetic processes under non-toxic chemicals are important for biocompatible application, particularly for additional integration of the nanoparticles with other biological substrates, which is useful in diagnostic procedures, drug delivery, therapies, and biomedical applications . Thus, a biocompatible protocol with a direct one-pot reaction in a mild condition and well-controlled shapes and sizes is needful in these fields. Many biomolecules such as liposomes , plant extracts , and chitosan [15, 16], as stabilizer and/or reducing agents, have been directly used to synthesize AuNPs. In the present investigation, we describe a new method of designing and synthesizing gold nanoparticles by using Tris(hydroxymethyl)aminomethane. As we have known, Tris is one of the most widely used buffers of nucleic acids and proteins in biochemistry and biotechnology, and has also been adopted as a ligand for the synthesis of chromatographic adsorbents [17, 18]. It is quite active in reduction reactions in various conditions due to its specific structure. More importantly, the reducing agent makes it possible to modify DNA during the process of preparing AuNPs. This feature is very useful for some applications, such as sensors, spectroscopic enhancers, quantum dot, nanostructure fabrication, microimaging methods, and ultrasensitive detection [19–22].
Gold nanoparticles were synthesized by Tris method. Chloroauric acid (AR; 97 ml, 0.01% m/m, SCRC, Beijing, China) with 4 ml Tris solution was stirred using a magnetic stirrer, reduced to Au(0) slowly at 600 rpm at 40°C, and remained for 10 min until it has no visual change for another 10 min. Then, 3 ml NaOH (pH > 14) solution was injected to the solution drop by drop, meanwhile increasing the temperature of the solution to 50°C slowly. We could see the color of the solution gradually change from pink to deep red wine color. After about 8 min, the temperature was decreased to room temperature while stirring was continued to cool the solution. The reaction solution was then centrifuged at 12,000 rpm for 20 min (Xiang Yi centrifugal machine, Changsha, China), and its supernatant was removed; then, the AuNP solution was diluted to the original concentration with ultrapure water (18.2 MΩ, produced by a Milli-Q system, Millipore Co., Billerica, MA, USA). Absorptions were measured using a UV-2450 spectrophotometer (Shimadzu Co., Nakagyo-ku, Kyoto, Japan) operated at a resolution of 1 nm. Atomic force microscopy imaging was performed on SPM-9600 (Shimadzu Co.). Samples for transmission electron microscope (TEM) analysis were prepared by dropping Au nanoparticle solutions onto carbon-coated copper grids. JEM-2100 F (accelerating voltage 200 kV, JEOL, Ltd., Akishima, Tokyo, Japan) was used for obtaining the TEM images, and dynamic light scattering (Nano-zeta-size 90, Malvern Instruments, Westborough, MA, USA) was used mainly for the measurement of particle size and zeta potential.
Results and discussion
UV-vis spectroscopy and atomic force microscopy
The images of transmission electron microscope
Dynamic light scattering (DLS) data of the size distributions of three kinds of gold nanoparticles
For DNA conjugate application, we can further optimize the experimental process to make the reaction conditions much more mildly and to keep the DNA intact under such environment. For this purpose, we replaced the magnetic stirring by ultrasonic vibration, and the heating temperature was set to 45°C. First, the chloroauric acid solution was treated by ultrasonic and heated for 15 min (actual temperature is about 30°C). Then, the mixed solution of 2 ml of 10 mM Tris containing DNA at a concentration of 1 ng/μl with 2 ml NaOH whose mass fraction is 1% was added, and then the solution was ultrasonically treated for one more hour. The final DNA gold nanoparticles were ready for further conjugation.
Zeta potential and mobility of pure DNA and gold nanoparticles prepared using Tris-DNA mixture at 25°C
Zeta potential (mV)
In summary, we presented a new biocompatible synthesis method of gold nanoparticles by Tris, a widely used buffer of nucleic acids and proteins. The method has a useful feature to allow modifying the DNA during the process of preparation of gold nanoparticles. However, the mechanism responsible for biomolecule-directed gold nanoparticle formation remains unclear due to the lack of structural information about biological systems and the fast kinetics of biomimetic chemical systems in solution .
Tris(hydroxymethyl)aminomethane, or 2-amino-2-(hydroxymethyl)-1,3-propanediol, coordinates with a number of metallic ions. In order to understand its unusual acid-base and redox activities, we added Tris solution to chloroauric acid solution until it is completely mixed with the gold ions. Then, we slowly heated the solution to 40°C while adding NaOH solution gently into the solution until the color is stable, immediately adjusting the temperature to room temperature by stirring the solution. In the whole process, temperature does not exceed 45°C, and the stirring speed is about 1,000 rpm or less. In this protocol, by using Tris in alkaline aqueous solution, we can prepare large Au nanoparticles with a polyhedral structure in a temperately process. DNA or cells in vitro are capable of maintaining their intrinsic characteristics under such mild conditions. By using ultrasonic vibration, we can even prepare one-pot DNA-gold nanoparticle conjugates directly, ready for further applications of sensing, imaging, and ultrasensitive detection in biomolecular field [27–29]. During the reaction, a high concentration of NaOH solution is used to promote the reaction rate.
This work was supported by the National Natural Science Foundation of China (No. 11274245) and Innovation Fund of Wenzhou University (31606036010187).
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