Etching of AuNPs Through Superoxide Radical Dismutation by Cu-Zn Superoxide Dismutase Resulted in Remarkable Changes of its Localized Surface Plasmon Resonance

Document Type : Research Paper

Authors

1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Medical Biotechnology, Applied Biophotonics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran

3 Center of Excellence in Electrochemistry, University of Tehran, Tehran, Iran

4 Biosensor Research Center, Endocrinology & Metabolism Molecular, Cellular Sciences Institute

5 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Abstract

Background: Superoxide dismutases (SODs) are categorized as antioxidant enzymes that are involved in many processes such as stress signalling responses and cell protection against free radical species. The primary function of SOD is the removal of produced radical species like superoxide ions in different physiological processes. There are various isozymes of SODs which are classified according to the metal cofactor in their active sites into four general types of Fe-SOD, Mn-SOD, Cu/Zn-SOD and Ni-SOD. Among metal nanoparticles, gold nanoparticles (AuNPs) are useful for biological purposes as sensing probe for determining critical analysis based on surface plasmon resonance and colorimetric method. In this study, the human Cu-Zn SOD expressed, purified, and its interaction with AuNPs based on a new colorimetric method was investigated.
Objectives: In this approach, a colorimetric detection method for SOD activity was developed based on the carboxylic stabilized AuNPs.
Material and Methods: The Ni-NTA Sepharose affinity column was performed for the purification process of enzyme. Following SOD purification, the enzyme activity in presence of AuNPs due to the possible etching in the presence of free radicals which are produced by riboflavin, methionine, Na2CO3 and potassium phosphate buffer, have been performed. In addition, Fluorescence spectroscopy analysis toward SOD and gold nanoparticle were performed.
Results: Superoxide radicals generated from the enzymatic reaction would preferentially etch AuNPs and resulted in remarkable changes of localized surface plasmon resonance of AuNPs, which is reduced in the presence of SOD. Under the optimized experimental conditions assay (pH~7.8 and 25 ˚C), better selectivity and sensitivity toward SOD activity was shown.
Conclusions: In this context, an indirect new colorimetric method for determining of SOD activity based on gold nanoparticles (AuNPs) was evaluated. According to the presented result, it may be concluded that by scavenging of free superoxide radicals in the presence of SOD, the amount of AuNP absorbance can be replenished.

Keywords

Main Subjects


1. Background

McCord and Fridorich discovered a copper protein known as Cu-Zn SOD that was dimmer and each of its monomeric units contain an active site, a copper ion, and a zinc ion, which was linked to histidine by imidazole ring. Copper acts as reactive metal and zinc ion is effective in pH stability ( 1 - 4 ). Superoxide dismutase (SOD, EC 1.15.1.1.) is a metalloenzyme and is considered an oxidoreductase enzyme. SODs have an essential role in the dismutation of superoxide radicals, which are cytotoxic agents to biomolecules such as proteins, DNA, and cells. These enzymes provide the first line of defense against ROS ( 3 , 5 ). Mutation in the SOD1 gene and subsequent aggregation of mutant proteins has been considered as one of the main influencing factors in the progress of Amyotrophic Lateral Sclerosis (ALS) disease ( 6 - 8 ).

Gold nanoparticles have a long history in biotechnology fields. Understanding the properties of new metal nanoparticles and discovering their potential uses are two main features in the wide variety of nanoscale synthesis ( 9 ). Many of the properties of nanomaterials are due to the surface-to-volume ratio and the spatial structure of electrons, photons and electrical fields inside and around particles. The features such as high activity, different elasticity, magnetic properties, increased conductivity or increased light reflection tendency, separate nanomaterials from other materials ( 5 , 9 ).

Gold nanoparticles have unique physicochemical properties that make them suitable tools for biological sensors, immune assays, targeted drug delivery, marking cells, molecular imaging, and medical applications ( 10 - 12 ). The size and shape of the AuNPs (spherical, rod, cage, cubic, etc.) can adjust their optical properties. Surface variations, shape, and size of spherical and spherical AuNPs have shown different results in terms of stability, bonding, and compatibility in the body ( 13 - 15 ).

The interaction of nanoparticles with biomolecules, especially proteins, is considered as the term of their bioactivity and creates a dynamic system that can have many benefits for both nanoparticles and proteins. The nanoparticle-protein bioconjugation may affect cell infiltration, inflammation, and also accumulation, degradation, and purification of nanoparticles ( 16 ). Understanding such interactions can lead to the production of environmentally friendly nanomaterials with surface-controlled properties in a biological environment ( 17 ).

The absorption of protein on the surface of nanoparticles is achieved by several forces such as hydrogen bonds, salvation van der Waals, and electrostatic interactions ( 18 ). The formation of nanoparticle-protein, also known as corona-protein, is a multistage process that depends not only on the properties of the nanoparticle but also on the interaction of proteins with the surrounding environment ( 17 ).

The binding of biochemical molecules to nanoparticles makes them useful for different applications and also makes the nanoparticle-protein complex more stable. In many cases, biological molecules, especially proteins, undergo structural changes in binding to the nanoparticles ( 17 ). Research on enzymes and their interaction with nanoparticles has been recently increased. Meanwhile, the interaction of superoxide dismutase enzyme and gold nanoparticle remains unknown. Currently, several methods have been reported for detection of the activity of SOD in association with AuNPs like the spectrofluorimetric method ( 19 ), mercury microelectrodes ( 20 ), and spectrophotometric method ( 21 ). However, these methods have their drawbacks, such as high cost and fluorescence quenching. So studies revealed that gold nanoparticle possesses strong localized surface plasmon resonance (LSPR) from the visible region to near-infrared region and high extinction coefficient ( 22 ). These properties are influenced by many factors such as size, shape, composition, and the surrounding media of the reaction.

In the current study, the SOD activity in presence of spherical AuNPs due to the possible etching in the presence of free radicals which are produced by riboflavin, methionine, Na2CO3 and potassium phosphate buffer, have been investigated. The novelty of the work relates to these aspects.

2. Objectives

Herein, after the expression and purification of human Cu-Zn superoxide dismutase, we proposed an effective procedure for colorimetric visualization of the enzyme through the etching of carboxylic-stabilized AuNPs.

3. Materials and Methods

3.1. Materials

K2HPO2, KH2PO4, CuSO4/ZnSO4, and Na2CO3 purchased from Merck (Germany). Ni-NTA Sepharose affinity column from Novagen, kanamycin, IPTG, Riboflavin, L-methionine, and NBT also from Sigma-Aldrich, was purchased. The water-soluble carboxylic-stabilized gold nanoparticles (AuNPs) with 1.6 nm used in this study were prepared.

3.2. Expression and Purification of Human Cu-Zn Superoxide Dismutase

The E. coli BL21 strain was transformed by human Cu-Zn superoxide dismutase coding gene-containing plasmids (pET28a plasmid) ( 6 ). Transformed bacteria were cultured in 10 mL LB medium with 50µg.mL-1 kanamycin and incubated overnight at 37 ˚C with shaking at 150 RPM. Then 1 mL of LB medium was added to 250 mL TB medium with kanamycin and incubated at 37 ˚C with 150 RPM shaking. After almost 3.5-4 h, 0.2 mM CuSO4 and 0.5 mM IPTG were added to TB medium, and incubation at 20 ˚C with150 RPM for 16h was performed. Then after the cell pellets were harvested by centrifugation at 6000 g for 10 min at 4 ˚C. The suspension was sonicated by lysis buffer (50 mM Tris-HCl PH 7.8). Next Centrifugation at 14000 g for 20 min, at 4 ˚C was done to collect the supernatant. Finally, purification with Ni-Sepharose column affinity chromatography was carried out. After that, the dialyzing under 100 mM sodium acetate, 10 mM EDTA and 100 mM NaCl (pH 3.8), 100mM sodium acetate and 200 µm ZnSo4, 200 µm CuSO4 (pH 5.5), 20 mM potassium phosphate (pH 7.4) buffers respectively were carried out. Then the concentration of the dialyzed extract was determined by Bradford assay using coomassie blue and bovine serum albumin as standard.

3.3. SOD Activity Assays

SOD activity was measured by a spectrophotometric method based on nitroblue tetrazolium (NBT) illumination method in 50 mM potassium phosphate buffer (pH 7.8) at 25 ˚C. One unit of SOD activity was defined as the amount of protein required to inhibit the rate of production of formazan by 50% under the assay conditions as described above. For measuring kinetic properties of the enzyme, different dilutions of Riboflavin (0.5, 0.25, 0.12, 0.06, and 0.03 µM) were prepared to determine the Km of SOD1 enzyme (from the original concentration of 20 µM).

L-Methionine (26 mM), Na2CO3 (50 mM), NBT (750 µM), and Riboflavin mixed, and each vial exposed to light after 15min and then the absorption at 560nm was read.

3.4. Fluorescence Spectroscopy of the Enzyme in the Presence of Gold Nanoparticles

Intrinsic fluorescence emission spectroscopy of enzymes in the presence of different concentrations of gold nanoparticles was measured by PerkinElmer LS-55. The excitation wavelength of human Cu-Zn SOD was 280 nm and the intrinsic emission spectra were recorded between 290 and 450 nm. All the fluorescence measurements carried out at 25 ˚C in the final volume of 60 microliters.

3.5. Investigating the Activity of the Enzyme in the Presence of Gold Nanoparticles in an Environment Containing Radical Superoxide

The enzyme activity was measured in the presence of gold nanoparticles (c=1×10-7 molar) and NBT. Different concentrations of SOD were prepared and then the equivalent molar of the enzyme was added to each solution. After incubation time (30 min), the activity of each assembly was measured by absorbance reading at 530 nm by PerkinElmer Lambda 25 UV-visible spectroscopy.

3.6. Investigation of the Absorption of Gold and Enzyme Nanoparticles in an Environment Free of Superoxide Radicals

In order to compare and verify the accuracy of our experiments in the previous section, only the different concentrations enzyme in the presence of a concentration of gold nanoparticles with 50 mM phosphate buffer at pH =7.8 evaluated, and the absorption of nanoparticles recorded at 530 nm by a reported Method ( 23 ).

4. Results

4.1. Cu-Zn Superoxide Expression

The expressed protein was purified based on the 6His-tagged fusion by affinity (Ni-NTA Sepharose) chromatography. SDS-PAGE analysis of the eluted enzyme showed more than 95% purity of the enzyme (Fig. 1).

Figure 1. SDS-PAGE of purified human Cu-Zn superoxide dismutase.

4.2. Determination of Kinetic Properties and Specific Activity of the Enzyme

To determine the kinetic properties and specific activity of the enzyme, the substrate was mixed and subjected to irradiation. Absorption, the rate of inhibition, and also its specific activity in the concentration of 0.1 mg.mL-1 of the enzyme were reported (Table 1). The parameters were determined according to the following formula. The inhibition was 78.8%, and its specific activity was 787 units/mg. Finally the kinetic values of the enzyme after drawing line Weaver-Burk plot (Fig. 2) showed that SOD1 enzyme was indicative of a relatively good enzyme tendency toward its substrate (Table 1).

Km (µM) Inhibition% Specific activity (units/mg protein) Vmax (unit/mg)
0.226 78.8 787 69.06
Table 1.Kinetic parameters of purified enzyme

Inhibition % = Δ(absorbance at 560 nm without enzyme) - Δ(absorbance at 560 nm with enzyme) × 100 Absorbance (reaction blank)Specific

Specific activity = % Inhibition Protein concentration (mg)

Figure 2. Michaelis Menten and Line Weaver Burke plots for the Kinetic Parameters of the enzyme for the riboflavin substrate

4.3. Fluorescence Spectroscopy

Intrinsic fluorescence measurement of the enzyme in the presence of different concentrations of gold nanoparticles was measured (Fig. 3), It was found that how the concentration of nanoparticles may affect the structure of the enzyme. The results showed that none of the nanoparticle concentrations had led to the exposure of inner aromatic residues to the solvent.

Figure 3. Structural analysis of the enzyme in the presence of different concentrations of Au-nanoparticles using intrinsic fluorescence emission of the enzyme. (a)-(f) shows intrinsic fluorescence of SOD in the presence of 0.5×10-7, 0.25×10-7, 0.12×10-7, 0.06×10-7, 0.03×10-7, 0.01×10-7 mol/lit of AuNP, respectively and (g) is the fluorescence emission of the enzyme alone.

4.4. Activity of Enzyme in the Presence of Gold Nanoparticles

The assay of the enzyme activity in the presence of superoxide radicals and nanoparticles was performed (Fig. 4). After the production of radical superoxide in the environment, gold nanoparticles began to etch, which occurs for metal nanoparticles in the presence of free radicals which affect the plasmon resonance of the surface. The results showed that the more free radicals in the environment, the higher the etching of AuNPs and thus the more decrease in the absorption.

Figure 4. Enzyme activity in the presence of Au-nanoparticles at various concentrations of the enzyme. (a) is the absorbance (at 530 nm) of free AuNP (b)-(f) is the absorption spectra of the same amount of AuNP in presence of constant concentration of superoxide radical generators and various amount of enzyme; 60, 30, 10, 7 and 3 µg/ml-1 of SOD1, respectively.

4.5. AuNp Absorbance Against SOD1 Concentration

The changes in the surface resonance of the gold nanoparticles were recorded, and the difference in their absorption was illustrated by a calibration curve of the various concentrations of superoxide dismutase enzyme. Therefore, given the relatively linear nature of the diagram showed that our results could lead to developing a method for measuring SOD1 enzyme activity (Fig. 5).

Figure 5. Absorption spectra of AuNP in the presence of superoxide radical source and various concentration of SOD1.

4.6. Absorption Spectra of Gold Nanoparticles and Enzyme without Superoxide Radical

Different concentrations of the enzyme without superoxide radicals in the environment placed in the vicinity of gold nanoparticles and no significant difference in absorptions were observed, and all of the spectra were overlapped (Fig. 6). This data showed that the absorbance reduction has only occurred in the presence of superoxide radical presumably through etching of AuNPs.

Figure 6. The absorption of AuNP in the presence of various concentrations of enzyme in the absence of superoxide radical with 50 mM potassium phosphate buffer at pH = 7.8.

5. Discussion

The application of nanomaterials in the biomedical sciences has been considered in recent decades. Spherical gold nanoparticles have found many applications because of their unique characteristics rather than bulk materials ( 24 - 26 ).

Gold nanoparticles can be attached to many biomolecules such as proteins, nucleic acids, and lipids for different practical purposes. These particles have unique properties such as high absorption, luminescence, and conductivity and even their usefulness to enhance electromagnetic reactions and provide them for sensing purposes. One of the most attractive aspects of gold nanoparticles is their application in GNPs-based assays by using colorimetric methods ( 27 - 30 ).

Superoxide dismutases are the primary protective barrier against the destructive effect of high concentrations of free radicals, especially superoxide, which cause genital mutations and cancer ( 31 ), and it is also accelerated in the aging process ( 32 , 33 ).

Detection based on colorimetric methods is considered the leading gold method for nanometric assay. Although, existing methods for these assays have still challenges such as nonspecific interactions with biomolecules and difficulties posed by in vivo applications ( 34 , 35 ). Colorimetric assays have a high potential for high-throughput applications because they are simple, inexpensive, and require minimal instrumentations. Colorimetric assays are very promising for their effectiveness in determining enzyme activity in the biological process. Besides they are excellent candidates for in vitro measurements ( 29 ).

Simin Lu et al. designed a colorimetric method for superoxide dismutase assay based on gold nanorods and observed that the activity of the enzyme could be kept ( 29 , 36 ). Zifen Deng et al. applied an approach in the direct transfer of electrons from copper and zinc superoxide dismutase to the surface of ZnO nanoparticles by stabilizing the enzyme on nanoparticles. The electrochemical results showed that the enzyme stabilization on the ZnO nanoparticle maintained its activity ( 37 ). Surin Hong et al. utilized superoxide dismutase to conjugate on AuNP surface (SOD1-AuNP) for detection of the structural evolution of SOD1 aggregates. The results indicated that significant changes in both surface plasmon resonance spectra and concomitant colors were observed. They showed that the gold nanoparticle (AuNP)-based highly sensitive and colorimetric detection of the temporal evolution of superoxide dismutase (SOD1) aggregates implicated in the pathology of amyotrophic lateral sclerosis (ALS) ( 38 ).

In another study by Reynaldo Villalonga et al., a bienzymatic supramolecular nanoassembly containing catalase and Cu, Zn-superoxide dismutase was performed. They concluded that Superoxide dismutase was 90-fold more resistant to inactivation by H2O2 after bienzymatic immobilization with catalase on metal nanoparticles ( 39 ).

Junwei Di et al. designed a superoxide dismutase (SOD) based biosensor via a silica sol-gel (SG) thin film and further immobilization of SOD and gold nanoparticles (GNPs) in silica SG network. The resulting biosensor exhibited a fast amperometric response to superoxide anion. Also, the biosensor exhibited high sensitivity and long-term stability ( 40 ).

There are not enough researches on the behavior of SOD and AuNPs to estimating of what effect these particles can have on the enzyme, for this purpose the interaction of human Cu-Zn superoxide dismutase with gold nanoparticles was studied through UV-visible spectroscopy and fluorescence techniques and proposed the colorimetric method for detecting the enzyme activity. In this study, the AuNPs aggregation was occurred due to surface attachment to SOD presumably through electrostatic interactions. This issue will reduce the initial tendency of SOD protein for aggregation as an amyloidogenic protein as indicated in recent similar studies on the aggregation of α-synuclein protein ( 41 - 44 ).

Kinetic properties of purified SOD are shown in (Table 1), which indicates the specific activity of the purified enzyme was 787 unit/mg. The kinetic parameters of the enzyme also showed that the activity of the purified enzyme was suitable for further analysis. Also, the fluorescence properties of enzymes in different concentrations of AuNPs were studied (Fig. 3).

In this work, we have developed an indirect new colorimetric method for determining SOD activity based on gold nanoparticles (AuNPs). For this purpose, a constant concentration of nanoparticles was placed adjacent to the produced superoxide radical and various concentrations of the enzyme which indicated that the lower the enzyme concentration, the less absorption of the nanoparticles (Fig. 5, 6). So we found that the higher the enzyme concentration will eliminate more free radicals. Therefore, the amount of etching of the nanoparticles is reduced, so the change in the surface plasmon resonance of the nanoparticles is also lower. However, based on the result presented in this manuscript it may be concluded that by scavenging free superoxide radicals in the presence of SOD, the amount of AuNP absorbance can be replenished (Fig. 7).

Figure 7. Schematic presentation of inhibition of AuNPs etching in presence of SOD1. PDBe ID for Superoxide dismutase is 5U9M (https://www.ebi.ac.uk/pdbe).

Acknowledgement

Financial support of this work was provided by Nooragen Pishroo company.

Conflict of Interest: The authors declare that they have no conflict of interest.

References

  1. Mann T, Keilin DJPotRSoLSB-BS. Haemocuprein and hepatocuprein, copper-protein compounds of blood and liver in mammals. Proc Royal So. B. 1938; 126(844):303-315. DOI
  2. Crapo JD, Oury T, Rabouille C, Slot JW, Chang LY. Copper, zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc Natl Acad Sci U S A. 1992; 89(21): 10405-10409.
  3. Khanna-Chopra R, Selote DS. Acclimation to drought stress generates oxidative stress tolerance in drought-resistant than-susceptible wheat cultivar under field conditions. EEB. 2007; 60(2):276-283. DOI
  4. Wang Y, Osatomi K, Nagatomo Y, Yoshida A, Hara K. Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthys olivaceus). Comp Biochem Physiol B. 2011; 158(4):289-296. DOI
  5. Zhang Z, Chen Z, Pan D, Chen L. Fenton-like reaction-mediated etching of gold nanorods for visual detection of Co(2+). Langmuir. 2015; 31(1):643-650. DOI
  6. Salehi M, Nikkhah M, Ghasemi A, Arab SS. Mitochondrial membrane disruption by aggregation products of ALS-causing superoxide dismutase-1 mutants. Int J Biol Macromol. 2015; 75:290-297. DOI
  7. Abbasabadi AO, Javanian A, Nikkhah M, Meratan AA, Ghiasi P, Nemat-Gorgani M. Disruption of mitochondrial membrane integrity induced by amyloid aggregates arising from variants of SOD1. Int J Biol Macromol. 2013; 61:212-217. DOI
  8. Ghiasi P, Hosseinkhani S, Noori A, Nafissi S, Khajeh K. Mitochondrial complex I deficiency and ATP/ADP ratio in lymphocytes of amyotrophic lateral sclerosis patients. Neurol Res. 2012; 34(3):297-303. DOI
  9. Djalali R, Chen Y-f, Matsui HJJotACS. Au nanowire fabrication from sequenced histidine-rich peptide. JACS. 2002; 124(46):13660-13661. DOI
  10. Ritchie CM, Johnsen KR, Kiser JR, Antoku Y, Dickson RM, Petty JTJTJoPCC. Ag nanocluster formation using a cytosine oligonucleotide template. J Phys Chem C. 2007; 111(1):175-181. DOI
  11. Saqib M, Qi L, Hui P, Nsabimana A, Halawa MI, Zhang W, et al. Development of luminol-N-hydroxyphthalimide chemiluminescence system for highly selective and sensitive detection of superoxide dismutase, uric acid and Co(2+). Biosens Bioelectron. 2018; 99:519-524. DOI
  12. Khamehchian S, Nikkhah M, Madani R, Hosseinkhani S. Enhanced and selective permeability of gold nanoparticles functionalized with cell penetrating peptide derived from maurocalcine animal toxin. J Biomed Mater Res Part A. 2016; 104(11):2693-2700. DOI
  13. Alex SA, Chakraborty D, Chandrasekaran N, Mukherjee AJRA. A comprehensive investigation of the differential interaction of human serum albumin with gold nanoparticles based on the variation in morphology and surface functionalization. RSC Advances. 2016; 6(58):52683-52694. DOI
  14. Jongjinakool S, Palasak K, Bousod N, Teepoo S. Gold Nanoparticles-based Colorimetric Sensor for Cysteine Detection. Energy Procedia. 2014; 56:10-18. DOI
  15. Strange RW, Antonyuk SV, Hough MA, Doucette PA, Valentine JS, Hasnain SS. Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes. J Mol Biol. 2006; 356(5):1152-1162. DOI
  16. Saptarshi SR, Duschl A, Lopata ALJJon. Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle. J Nano Biotechnol. 2013; 11(1):26. DOI
  17. Wangoo N, Suri CR, Shekhawat GJAPL. Interaction of gold nanoparticles with protein: a spectroscopic study to monitor protein conformational changes. Appl Phys Lett. 2008; 92(13):133104. DOI
  18. Bannister W, Bannister J, Barra D, Bond J, Bossa F. Evolutionary aspects of superoxide dismutase: the copper/zinc enzyme. Free radical research communications. Free Radic Res. 1991; 12(1):349-361. DOI
  19. Tang B, Wang Y, Chen Z-zJSAPAM, Spectroscopy B. Catalytic spectrofluorimetric determination of superoxide anion radical and superoxide dismutase activity using N, N-dimethylaniline as the substrate for horseradish peroxidase (HRP). Spectrochim Acta A Mol Biomol Spectrosc. 2002; 58(12):2557-2562. DOI
  20. Daniele S, Orsega EF, Marson G, Argese EJJoEC. Copper, zinc superoxide dismutase activity evaluation using mercury microelectrodes. J Electroanal Chem. 2000; 492(2):112-119. DOI
  21. Nebot C, Moutet M, Huet P, Xu J-Z, Yadan J, Chaudiere JJAb. Spectrophotometric assay of superoxide dismutase activity based on the activated autoxidation of a tetracyclic catechol. Anal Biochem. 1993; 214(2):442-451. DOI
  22. Keighron JD, Keating CDJL. Enzyme: nanoparticle bioconjugates with two sequential enzymes: stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles. Langmuir. 2010; 26(24):18992-9000. DOI
  23. Mondal B, Kamatham N, Samanta SR, Jagadesan P, He J, Ramamurthy VJL. Synthesis, characterization, guest inclusion, and photophysical studies of gold nanoparticles stabilized with carboxylic acid groups of organic cavitands. Langmuir. 2013; 29(41):12703-12709. DOI
  24. Doria G, Conde J, Veigas B, Giestas L, Almeida C, Assunção M, et al. Noble metal nanoparticles for biosensing applications. Sensors. 2012; 12(2):1657-1687. DOI
  25. Bai X, Shao C, Han X, Li Y, Guan Y, Deng ZJB, et al. Visual detection of sub-femtomole DNA by a gold nanoparticle seeded homogeneous reduction assay: toward a generalized sensitivity-enhancing strategy. Biosens Bioelectron. 2010; 25(8):1984-1988. DOI
  26. Wu. Fluorescent detection of lead in environmental water and urine samples using enzyme mimics of catechin-synthesized Au nanoparticles. ACS Appl Mater Interfaces. 2013; 5(4):1503-1509. DOI
  27. Zhen Z, Tang L-J, Long H, Jiang J-HJAc. Enzymatic immuno-assembly of gold nanoparticles for visualized activity screening of histone-modifying enzymes. Anal Chem. 2012; 84(8):3614-3620. DOI
  28. Tan LT. Filamentous tropical marine cyanobacteria: a rich source of natural products for anticancer drug discovery. J Appl Phycol. 2010; 22(5):659-676. DOI
  29. Hutter E, Maysinger DJTips. Gold-nanoparticle-based biosensors for detection of enzyme activity. J Pharmacol Sci. 2013; 34(9):497-507. DOI
  30. Oishi J, Han X, Kang JH, Asami Y, Mori T, Niidome T, et al. High-throughput colorimetric detection of tyrosine kinase inhibitors based on the aggregation of gold nanoparticles. Anal Biochem. 2008; 373(1):161-163. DOI
  31. Oberley LW, Buettner GRJCr. Role of superoxide dismutase in cancer: a review. Cancer Res. 1979; 39(4):1141-1149.
  32. Li Y, Huang T-T, Carlson EJ, Melov S, Ursell PC, Olson JL, et al. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet. 1995; 11(4):376. DOI
  33. Niyomploy P, Srisomsap C, Chokchaichamnankit D, Vinayavekhin N, Karnchanatat A, Sangvanich P. Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms. J Pharm Biomed Anal. 2014; 90:72-77. DOI
  34. Albizu L, Cottet M, Kralikova M, Stoev S, Seyer R, Brabet I, et al. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol. 2010; 6(8):587. DOI
  35. Mayilo S, Kloster MA, Wunderlich M, Lutich A, Klar TA, Nichtl A, et al. Long-range fluorescence quenching by gold nanoparticles in a sandwich immunoassay for cardiac troponin T. Nano Lett. 2009; 9(12):4558-4563. DOI
  36. Lu S, Zhang X, Chen L, Yang P. Colorimetric determination of ferrous ion via morphology transition of gold nanorods. Microchim Acta. 2017; 185(1):76. DOI
  37. Deng Z, Tian Y, Yin X, Rui Q, Liu H, Luo Y. Physical vapor deposited zinc oxide nanoparticles for direct electron transfer of superoxide dismutase. Electrochem Commun. 2008; 10(5):818-820. DOI
  38. Hong S, Choi I, Lee S, Yang YI, Kang T, Yi J. Sensitive and colorimetric detection of the structural evolution of superoxide dismutase with gold nanoparticles. Anal Chem. 2009; 81(4):1378-1382. DOI
  39. Villalonga R, Cao R, Fragoso A, Damiao AE, Ortiz PD, Caballero J. Supramolecular assembly of β-cyclodextrin-modified gold nanoparticles and Cu, Zn-superoxide dismutase on catalase. J Mol Catal B Enzym. 2005; 35(4-6):79-85. DOI
  40. Di J, Peng S, Shen C, Gao Y, Tu Y. One-step method embedding superoxide dismutase and gold nanoparticles in silica sol–gel network in the presence of cysteine for construction of third-generation biosensor. Biosens. Bioelectron 2007; 23(1):88-94. DOI
  41. Salehi M, Nikkhah M, Mohammadi S, Hosseinkhani S. Transient transfection of WT-αS and A53T-αS brought about a mild apoptosis due to degradation of released cytochrome c through PARC. Int J Biol Macromol. 2020. DOI
  42. Mohammadi S, Nikkhah M, Hosseinkhani S. Loss in Toxic Function of Aggregates of α-Synuclein Mutants by a β-Synuclein Derived Peptide. Protein Pept Lett. 2017; 24(8):757-764. DOI
  43. TiO2 nanoparticles as potential promoting agents of fibrillation of α-synuclein, a parkinson’s disease-related protein. Iran J Biotechnol. 2017; 15(2):87. DOI
  44. Mohammadi S, Nikkhah M, Hosseinkhani S. Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor. Int J Nanomed. 2017; 12:8831. DOI