550 600 650 700 750 800 850
0.0
Figure S1. Emission spectra of [Ru(bpy)2(im)2]2+ with various concentrations of Rua63+.
550 600 650 700 750 800 850
0.0
Figure S2. Emission spectra of [Ru((CH3)2bpy)2(im)2]2+ with various concentrations of Rua63+.
0.000 0.002 0.004 0.006 0.008 0.010 1.0
1.2 1.4 1.6
I I
o[Q], M Y = 0.95691 + 59.8957 * X R = 0.98929
Figure S3. The Stern-Volmer plot of emission intensity of [Ru(bpy)2(im)2]2+ with Rua63+.
0.000 0.002 0.004 0.006 0.008 0.010 1.0
1.5 2.0
o[Q], M Y = 1.00712 + 95.96381 * X R = 0.99983
Figure S4. The Stern-Volmer plot of emission lifetime of [Ru(bpy)2(im)2]2+ with Rua63+.
0.000 0.002 0.004 0.006 0.008 0.010 1.0
1.2 1.4
I I
o[Q], M Y = 0.97027 + 48.77246 * X R = 0.9925
Figure S5. The Stern-Volmer plot of emission intensity of [Ru((CH3)2bpy)2(im)2]2+
with Rua63+.
0.000 0.002 0.004 0.006 0.008 0.010 1.0
1.2 1.4 1.6
o[Q], M Y = 1.00487 + 64.42619 * X R = 0.99988
Figure S6. The Stern-Volmer plot of emission lifetime of [Ru((CH3)2bpy)2(im)2]2+
with Rua63+.
0.000 0.002 0.004 0.006 0.008 0.010 1
2 3 4 5
I I
o[Q], M Y = 0.91755 + 417.55925 * X R = 0.99906
Figure S7. The Stern-Volmer plot of emission intensity of [Ru((COO−)2bpy)2(im)2]2−
with Rua63+.
0.000 0.002 0.004 0.006 0.008 0.010 1
2 3 4 5
o[Q], M Y = 1.08199 + 320.28486 * X R = 0.99589
Figure S8. The Stern-Volmer plot of emission lifetime of [Ru((COO−)2bpy)2(im)2]2−
with Rua63+.
0.000 0.002 0.004 0.006 0.008 0.010 1.0
1.5 2.0 2.5
Y = 1.04211 + 146.5077 * X R = 0.99659
o[Q], M
Figure S9. The Stern-Volmer plot of emission lifetime of [Ru(phen)2(im)2]2+ with Rua63+.
Table S1. Supplement for the bimolecular quenching reaction between ruthenium modified Fe3+-cyt c and Rua63+.
kobs, s-1 kin, s-1
[Q], mM kq, s-1M-1 KSV, M-1 QYexdm-Fe3+-cyt c 2.10 x 107 1.99 x 107 5.13 2.14 x 108 11 5.2%
bpy-Fe3+-cyt c 1.79 x 107 1.47 x 107 5.29 6.05 x 108 41 17.9%
dc-Fe3+-cyt c 3.37 x 107 1.57 x 107 5.35 3.36 x 109 214 53.4%
Table S2. Supplement for the bimolecular quenching reaction between ruthenium modified Fe2+-cyt c and Rua63+.
kobs, s-1 kin, s-1
[Q], mM kq, s-1M-1 KSV, M-1 QYexdm-Fe2+-cyt c 2.39 x 107 1.99 x 107 5.33 7.50 x 108 38 16.7%
bpy-Fe2+-cyt c 2.01 x 107 1.47 x 107 5.20 1.04 x 109 71 26.9%
dc-Fe2+-cyt c 3.23 x 107 1.57 x 107 5.35 3.10 x 109 197 51.4%
Chapter 2
Photophysical properties of Ru(bpy) 2 dppz 2+ and ethidium bromide in microenvironment of micelle
and their application for determination of SDS
concentration
Abstract
Sodium dodecyl sulfate (SDS) is the most common detergent and it has been widely utilized in biomolecules separation. However high percentages of SDS mix with the analyte would interfere with the mass spectrum identification. Therefore, quick evaluation of the precise concentration of the residual SDS especially at low concentration is important to assure accurate mass measurement. Inorganic complex, Ru(bpy)2dppz2+, was utilized as the sensor for its famous light switch mechanism. As the concentration of SDS increases, the emission intensity of Ru(bpy)2dppz2+
increases. However, at the attempt to lower the SDS concentration below 0.1%, Ru(bpy)2dppz2+ precipitates out. An organic chromophore, ethidium bromide (EtdBr), was employed to detect low concentration of SDS. Both absorption and emission spectra of EtdBr changed in various concentration of SDS. In the low concentration of SDS (0−0.1%), formation of EtdBr-SDS complex results a large red shift of
absorption spectra. The ion pairs also cause the static quench of the EtdBr excited state, therefore, the emission intensity decreases. While the concentration of SDS is above 0.1%, SDS starts aggregating to form micelle which protects EtdBr from proton quenching and emission intensity is recovering. At above the CMC of SDS, the
emission intensity remains unchanged and is higher than that without SDS. The assay for determination of SDS concentration by EtdBr has been proposed. Despite the suitable range is small, the lower limit is around the range of no mass interference.
Introduction
Proteomics is a large-scale study of proteins from cells which is focused on their expressions and interactions. Though human genome is estimated to contain
20000−25000 genes, after transcription, translation and post-translational
modifications, the human proteome is estimated to comprise more than one million proteins. Considering all the structures and properties, proteomics is far more
complicated than genomics. The most widely applied technologies in proteomics are 2D gel electrophoresis and mass spectroscopy. As mass spectroscopy continues to develop advance methods for sequencing and identifying proteins,1-6 gel
electrophoresis is routinely employed for protein purification. Gel electrophoreses separate complex protein mixtures and nucleic acids based on their size or charge. As the method evolves, 2D gel electrophoresis is now an important technique for high resolution profiling of low abundance proteins.7 The first dimension is usually performed on pH gradients. Each protein will migrate to its isoelectric point (pI), the point at which the net charge of protein is zero. The process is called isoelectric focusing and the separation is according to charge. The second dimension is
performed on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the separation is according to the molecular weight. 2D gel delivers a proteomic map containing massive information. Whole protein directly from gel or in-gel digestion is becoming the standard sample preparation for the mass spectroscopic identification of proteins in the course of proteomic analysis.8,9 However high percentages of SDS mix with the analyte would interfere with the mass spectrum identification. The anionic surfactant, SDS, is the most common active ingredient in detergents to clean the greasy stain. It has also been widely utilized in biological assays such as denaturation in proteomics and lysing cells during DNA extraction.
SDS can denature the proteins by disrupting their non-covalent bonds and, therefore, destroys their native structure (conformation). Although the analyte can be desalted by passing through a reverse-phase C18 column to remove most of the SDS, small
amounts of SDS remain. The residual SDS may bind with the analyte and results in the deviation of mass-to-charge ratio (m/z).10-13 Therefore quick evaluation of the precise concentration of the residual SDS especially at low concentration is important to make sure accurate mass measurement.
For fast and convenient measurement, absorption and luminescence
spectroscopies are applied. In general, luminescence spectroscopy is more sensitive than absorption spectroscopy. Luminescence spectrum is against a dark background, therefore, small emission intensity will reveal striking feature. Several organic chromophores have been studied in the surfactant aqueous solution.14,15 Surfactants are usually amphiphilic organic compounds that contain both hydrophobic and hydrophilic functional groups. When added into water, single tail amphiphiles form micelles in solution and monolayers on the surface of the water, while some
monomers remain in solution. Double tails amphiphiles form bilayers instead of micelles. Critical micelle concentration (CMC) is an important feature for micelle formation. When the surfactant concentration is above CMC, surfactant aggregates to form normal micelle with the hydrophilic heads in contact with surrounding water and embeds the hydrophobic single tails in the micelle center. The various interactions between surfactant and water solvent demonstrate that solvatochromic effect may be found for the organic chromophore.16 Therefore they are potential candidates for detecting the concentration of SDS.
For example, an organic compound, ethidium bromide (EtdBr, Figure 1a), is studied here. EtdBr had been used in veterinary medicine to treat cattle for
trypanosomosis, a disease caused by trypanosomes since the 1950s.17 However the
high incidence of antibiotic resistance makes this treatment impractical in some areas.
In the present day, EtdBr is commonly used as nuclei acid stain for PAGE or agarose gel electrophoresis in molecular biology. Although EtdBr is suspected as mutagen or carcinogen depends on the dose of the exposure, it is best known for its DNA
intercalation ability duo to the planar ring system. Impressively, when EtdBr intercalates into double strand DNA the emission intensity increases about ten-fold compare to that in pure water.18 The amine group may be protected by the
hydrophobic base pairs which reduces the possibility of proton quench. Due to the giant increase of emission intensity, EtdBr has been widely used as DNA sensor.19,20 Besides, the absorption and fluorescence spectra of EtdBr are solvent dependence indicates the existence of solvatochromic effect.21 Based on these properties it is feasible to use EtdBr to evaluate the concentration of SDS.
Figure 1. Structure of (a) EtdBr and (b) Ru(bpy)2dppz2+.
Despite EtdBr can be utilized to measure the SDS concentration, it has weak emission in the aqueous solution. Therefore, the light on effect is not significant. To get better resolution, an inorganic complex is utilized. Ruthenium complex,
Ru(bpy)2dppz2+ (bpy = 2,2’-bipyridine, dppz = dipyrido[3,2-a:2’,3’-c]phenazine, Figure 1b) is stable, inert and water soluble. It has a unique photophysical property
that emission is strong in organic solvents or aprotic solvents but is absent in pure water or protic solvents. This behavior is due to the lone pair electrons on the nitrogen atoms of dppz interact with protic solvent. Upon excitation, the excited state of
Ru(bpy)2dppz2+ undergoes vibrational relaxation to the ground state through N−H stretching. In addition, though no phosphorescence in aqueous solution, the addition of DNA restores the emission. This behavior is the so-called light switch
mechanism.22 From X-ray structure analysis of DNA, the well-known double helix backbone is built from specific hydrogen bond of adenine-thymine and
guanine-cytosine pairs.23 The space between each base pair is about 3.4 Å . The layer by layer stacking base pairs of DNA produce an organized environment so that dppz ligand can intercalate into the base pairs and be stabilized by the − stacking.24,25 The DNA surrounding will avoid the interaction between the lone pair electrons on the nitrogen atoms of dppz to water, therefore, blocks the proton quench. This property makes Ru(bpy)2dppz2+ an ideal probe for DNA. The unusual emission enhancement of Ru(bpy)2dppz2+ has been applied in many fields such as study the process of photoinduced charge transfer through DNA.26,27 Similarly, the compact structure of normal micelle formed from surfactants produces a protected area from solvent, and the microheterogeneous environments of DNA and micelle both contain a negatively charged surface and hydrophobic interior. It implies the occurrence of emission enhancement for Ru(bpy)2dppz2+ in surrounding of DNA or micelle.28 Even though the reactions between molecules bound to DNA or micelle demonstrate striking differences which can be correlated with the distinct character of the highly ordered, − stacking base pairs in DNA in contrast to the disordered, aliphatic chains in the micelle, the immense structures of DNA and micelle are served as the sluggish solvent around Ru(bpy)2dppz2+ which will greatly reduce the proton quench and gives higher emission intensity. Based on the remarkable light switch mechanism,
Ru(bpy)2dppz2+ had been utilized to detect the concentration of SDS.
In this chapter, the photophysical properties of Ru(bpy)2dppz2+ in three
surfactant aqueous solution; SDS, TX-100 and CTAB, were investigated. In addition, the photophysical properties of EtdBr in the microenvironment of SDS, TX-100 and CTAB solution were also studied despite it had been reported previously.29 Since earlier report was performed in concentrations close to CMC, the interaction between EtdBr and surfactants at concentration below CMC was less mentioned. The sharp emission increase in SDS aqueous solution has made Ru(bpy)2dppz2+ and EtdBr potential candidates for SDS assay reagents.
Experimental Section
Materials
Sodium dodecyl sulfate (SDS, 98.5%) and sodium hexyl sulfate were purchased form Aldrich. Ethidium bromide (EtdBr, 95%), cetyltrimethylammonium bromide (CTAB, 99%), sodium sulfate (99%), sodium methyl sulfate (99%) and dimethyl sulfoxide (DMSO) were obtained from Acros. Triton X-100 (TX-100), acetonitrile (CH3CN) and dichloromethane (DCM) were purchased from J. T. Baker. Sodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) were obtained from Merck. Trifluoroacetic acid (TFA, 99%) was purchased from Riedel-de Haën. Tetrabutylammonium hydroxide (TBAOH, 40% in water, ~1.5 M) was obtained from Fluka. Sodium octyl sulfate (99%) and sodium tetradecyl sulfate (95%) were obtained from Alfa Aesar. All chemicals were used as received. Ru(bpy)2dppz2+ was synthesized according to the literature method.30