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Spring 5-25-2015 Terpyridine Functionalized Dye-Doped Silica Nanoparticles (DDSN) for Detection of Metal Ions Jonathan J. Liu University of San Francisco, [email protected]
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Recommended Citation Liu, Jonathan J., "Terpyridine Functionalized Dye-Doped Silica Nanoparticles (DDSN) for Detection of Metal Ions" (2015). Master's Theses. 216. https://repository.usfca.edu/thes/216
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Terpyridine Functionalized Dye-Dope Silica Nanoparticles (DDSN) for Detection of Metal Ions
Jonathan J Liu Thesis Advisor: Lawrence D. Margerum University of San Francisco
Terpyridine Functionalized Dye-Doped Silica Nanoparticles (DDSN) for Detection of Metal Ions
Thesis Written by Jonathan J Liu
This Thesis is written under the guidance of the faculty advisory committee, and approved by all its members, has been accepted in partial fulfillment of the requirements for the degree of
Master of Science in Chemistry at the University of San Francisco
Thesis Committee
Lawrence Margerum, Ph.D. Research Advisor
Jeff C. Curtis, Ph.D. Professor
William Melaugh, Ph.D. Professor
Marcelo Camperi, Ph.D. Dean, College of Arts and Sciences Contents Chapter 1 1 Introduction 1 1.1 The Properties and Applications of Silica Nanoparticles ...... 1 1.2 Fluorescence Basics and Quenching Theories ...... 6 1.3 Terpyridine as a Method Ion Binding Receptor ...... 13 1.4 Metal Ion Sensing ...... 16 1.5 Turn-on Sensors ...... 21 Chapter 2: Experimental ...... 37 2.1 Materials ...... 37 2.2 Instrumentation ...... 37 2.3 Preparation of 1-(5-([2,2’:6’,2’’-terpyrdin]-4’-yloxy)pentyl)-3-(3-(ethoxylsilyl)propyl)urea (Terpyridine Silane) ...... 40 2.4 Synthesis of DDSN with terpy and amine surfaces ...... 41 2.6 Determination of FITC Loading (mol/mg) on TAD-SNP ...... 43 2.7 Determination of TAD-SNP Solubility and Stability ...... 43 2.8 Metal Ion Quenching Assay (Turn-off Sensing)2 ...... 43 2.9 Quencher Disruption Assay(Turn-on Sensing)3 ...... 43 3. Fluorescence Quenching via Metal Ion Binding to TAD-SNP 45 3.1 Introduction ...... 45 3.2 Characterization of DDSNP ...... 47 3.2.1 Dye Loading on DDSNP ...... 47 3.2.2 Visual Terpyridine Test for SNP Dispersions ...... 47 3.2.3 UV-Vis Characterization of DDSNP ...... 48 3.2.4 Fluorescence Spectra of Dyes on DDSNP ...... 48 3.2.5 Transmission Electron Microscopy Images of DDSNP ...... 48 3 Future Work ...... 67 4. Quencher Displacement Assays (QDA) and Fluorescence Recovery with Metal Ion Chelated Dye Doped Terpyridine Nanoparticles 70 4.1 Introduction ...... 70 4.2 Results and Discussion ...... 71 4.3 Future Work ...... 75
iii Table of Tables Table 3- 1. Nanoparticle and dye concentration. Values determined from dye loading assay and gravimetric nano- particle analysis post drying of nanoparticle solution………………………………………………………… 47 n+ Table 3- 2. % Quenching of initial intensity, I0/I, @ 320 nm TD-SNP and 300 µM M ………...…………...51 Table 3- 3. Stability Constants for Metal Bound Terpyridine………………………………………………….51 2 Table 3- 4. Tabulated Stern-Volmer constants, τ0KSV, and regression fits, R for FITC-Terpy-NP1 versus metal nitrate solutions; …………………………………..……………………………………………………………60 Table 3- 5.Stern-Volmer constants for RITC-NP1 fluorescence quenching with metal ions………..65
iv Table of Figures
Figure 3- 1 Schematic representations of DDSNP surfaces for TD-SNP, TAF-SNP, and TyAzRx-SNP ...... 47 Figure 3- 2. TEM Photos of FITC-Terpy-NP1 from UC-Berkeley Electron Microscope Lab (scale unit: 50 nm) .... 49 Figure 3- 3. TEM Photos of FITC-Terpy-NP1 from UC-Berkeley Electron Microscope Lab (scale unit: 0.2 µm) ... 50 Figure 3- 4. Scheme of Fluorescence Quenching upon Introduction of Metal Ions ...... 51
Figure 3- 5 Fluorescein propyl triethoxysilane; λex: 490 nm; λem: 525 nm ...... 53 Figure 3- 6 Overlay plot of FITC-terpyridine-NP1 (TAF-NP) fluorescence quenching in the presence of increasing amounts of Fe3+ (1:1 ethanol:ACN; 80µg/ml nanoparticle) ...... 54 Figure 3- 7 Overlay plot of FITC-terpyridine-NP1 (TAF-NP) fluorescence quenching in the presence of Cu2+ (1:1 eth- anol:ACN; 80µg/ml nanoparticle) ...... 55 Figure 3- 8 Overlay plot of FITC-terpyridine-NP1 (TAF-NP) fluorescence quenching in the presence of Fe2+ (1:1 etha- nol:ACN; 80µg/ml nanoparticle) ...... 55 Figure 3- 9 Quenching of nanoparticle, FITC-terpyridine-NP1, fluorescence under the presence of Zn2+, spectral over- lap (solvent: 1:1 ethanol:ACN; 80µg/ml nanoparticle concentration) ...... 56 Figure 3- 10 FITC-Terpy-NP1 quenching sensitivity versus metal ions at a range of concentrations. The nitrate salts were used for all of the metal ions except Fe3+ where the chloride salt was used. (20 µg/ml DDSNP solution; 1:1 Etha- nol:ACN) ...... 57 Figure 3- 11 Stern-Volmer plot of FITC-Terpy-NP1 versus metal concentration for [Zn2+], [Ni2+],[ Fe3+], and [Pb2+]; (20 µg/mL DDSN solution in 1:1 Ethanol:ACN) ...... 58 Figure 3- 12 Stern-Volmer plot of FITC-Terpy-NP1 versus metal concentration for [Zn2+], [Co2+], and [Cu2+]; (20 µg/ mL DDSN solution in 1:1 Ethanol:ACN) ...... 58 Figure 3- 13 Polynomial Fit for Stern-Volmer plot of FITC-Terpy-NP1 versus metal concentration for [Cu2+] and [Co2+] ...... 59 Figure 3- 14 Fluorescence quenching of RITC-Terpy-NP upon the introduction of Ni2+ up to 3 µM. (20 µg/ml DDSNP; in EtOH) ...... 62 Figure 3- 15 Fluorescence quenching of RITC-Terpy-NP upon the introduction of Cu2+ up to 3 µM. (20 µg/ml DDSNP; in EtOH) ...... 63 Figure 3- 17 Fluorescence quenching with RITC-Terpy-NP1. Metal ion solutions were made from metal nitrates. (20 µg/ml DDSNP; in EtOH; λex = 525 nm, λem = 575 nm) ...... 63 Figure 3- 18 Stern-Volmer plot of RITC-Terpy-NP1 versus metal concentration for [Zn2+], [Fe3+], and [Pb2+]; (20 µg/ mL DDSNP in Ethanol) ...... 64 Figure 3- 19 Stern-Volmer plot of RITC-Terpy-NP1 versus metal concentration for [Zn2+], and [Ni2+] (20 µg/mL DDSNP in Ethanol) ...... 64 Figure 3- 20 Stern-Volmer plot of RITC-Terpy-NP1 versus metal concentration for [Zn2+], [Cu2+], and [Co2+]; (20 µg/ mL DDSNP in Ethanol) ...... 65
Figure 4- 1 Diagram for our quencher displacement assay surface ...... 70 Figure 4- 2. Effect of addition of five equivalents of substrate to a metal ion quenched terpy-DDSN...... 72 Figure 4- 3 Lewis Structure of the 5 tested substrates...... 72 Figure 4- 4 Effect of substrate on emission recovery for FITC-NP1-Cu2+ (20 µg/ml NP1; 25 µM Cu2+) ...... 73 Figure 4- 5 Effect of L-histidine on emission recovery for Cu2+-FITC-terpy-NP1 (20 µg/ml NP1; 2.5 µM Cu2+) .... 74
v Abstract In an effort to synthesize a novel silica nanoparticle platform for the fluorescent sensing of metal ions and future use in QDA, we synthesized from the bottom up a novel 2, 2’: 2’, 6- terpyridine functionalized fluorescein doped silica nanoparticle system capable of the sensitive detection of Cu2+ and Co2+. To further probe the properties of our nanoparticle system, we substitute the fluorescein dye of rhodamine and have observed differences in the selectivity and sensitivity of the system indicative of the potential customizability of the system. Initial QDA results on the fluorescein system suggest that while the signal is returned upon the introduction of EDTA, L-histidine, and epinephrine, the fluorescein dye in our system may interact with many of the anions tested.
Chapter 1
Introduction 1.1 The Properties and Applications of Silica Nanoparticles Research into nanoparticles is increasing due in part to the curious properties nanoparticles possess due to their size. 1-3 Nanoparticles, defined as being in the range between 1-99 nm, are unique in that they, as opposed to single molecules or bulk material, tend to undergo rapid Brownian motion when dispersed in a solution and thus stay suspended at low concentrations. 2-4 As the particle size is smaller than the wavelength of visible light, the suspension remains transparent despite bulk materials of the same type reflecting or refracting light at larger particle sizes. 1-6 Nanoparticles are of particular interest to analytical chemists and bioanalytical chemists because of their potential for use as benign sensors or drug delivery systems. Due to their small size, nanoparticles can pass through the renal barrier unobstructed leading to safe disposal from the body. The wide range of surface modification possibility then provides sufficient customization to target potential antigens.2, 7-14
Silica nanoparticles (NPs) are receiving particular interest due to the rich chemistry available to modify the surfaces and the relative cheapness and ease of synthesis compared to gold or iron NPs respectively.12, 15-22 Synthesis of silica particles can follow either the top down or the bottom up approach, but the former is typically reserved for micron sized particles and involves high energy mechanical milling.23, 24 However, mechanical milling can only be effective and efficient up to a point, as the grain required becomes smaller, more energy and time is required to bring down the median radius of the glass beads.23 In 1968, Dr. Werner Stöber, building off of work by Dr. Kolbe, published a study on the synthesis of colloidal silica particles of single micron sized silica nanoparticles through the use of tetraorthosilane otherwise known as tetraethoxy orthosilane (TEOS) monomers reacting in a solution of ethanol with an excess of water and ammonia. Their research demonstrated the ability to control size distribution by altering the relative ratios of the silane monomers, water, and ammonia and incubation time.25 Although the study had initially only focused on generating micron sized silica colloids, further research on the topic has yielded the ability for the method to synthesize nanometer sized silica nanoparticles.20, 22, 26-29
The exact mechanism for nanoparticle formation is still under debate but experiments in microgravity and small-angle x-ray scattering experiments suggest that nanoparticle formation and growth is through fast nucleation concurrent with controlled monomer aggregation increasing both nanoparticle concentration and size over time.24, 30, 31 Additionally, the synthesis of the particles can be carried out in a variety of pH environments utilizing different acids and bases as reaction catalysts.22, 24, 32 The pH of the solution and the nature of the catalyst influences the rate of particle formation and particle structure leading to a robust systems with a large amount of synthetic control.25, 29, 33
Commercial silica nanoparticles can be synthesized using monomers or through the industrial process of seeded water glass.24, 25, 29 Water glass, or sodium silicate, is formed by mixing silica with sodium carbonate under high heat and pressure.24 The resulting mixture is typically synthesized and dissolved into aqueous environments resulting in a high viscosity clear liquid.24 Water glass is typically used in cements, fire protection, and textile industries, but the silica can be nucleated under the right conditions to form nanoparticles without additional acid or base introduction. 29 Industrial synthesis of silica nanoparticles is typically through this route as it is cheaper than monomer condensation, despite requiring high temperatures and pressures.24
Silica nanoparticles form via monomer condensation processes through silane chemistry and the hydration of one of the ethoxy arms of TEOS.24, 25 Acid catalyzed reactions and base catalyzed reactions are considered different during nucleation phase as they are believed to progress through slightly different mechanisms based on pH, the acid catalyzed reactions are faster and favor chain formation, while catalysis under basic conditions is believed favor the total hydrolysis of the silane monomer prior to nucleation.24 A study done on the optimal pH for silica gel formation at high silane monomer concentration determined that silica was stable and tended to form nanoparticles around the pH of 5.5 –
2
9.0 and would gel if straying in either direction.25 Despite this distinction, at low monomer concentrations, there is no measureable difference between acid and base catalyzed react products.34-36
Additionally, at high concentrations with a suitable salt, the acid pathway is able to create gels, thus emphasizing the importance of solvent composition during nanoparticle synthesis.24
In the work to be reported here, we chose to proceed through the base catalyzed pathway for silica nanoparticle formation (see Fig. 1-1) due the improved stability over the acid pathway even in the presence of unanticipated salts.
Figure 1- 1. Synthetic Scheme of TEOS hydration and monomer formation used in our work
Surface modification is an essential part to making silica particles useful. Although silica could theoretically bind oxygen or nitrogen groups in organic molecules or bind to metal ions through its oxygen groups, the overall structure of the silica crystal will be overall positive or negative and the strain on the crystal structure would therefore make it unfavorable.24 Compounds that bind directly to the silica crystal are compounds that will typically increase the dissolution rate of silica such as HF or strong mineral acids.24 Of particular note are catechol compounds or catechol derivatives. Research done in the
1960s showed that catechol compounds will weakly bind to silica particles and to surface silyl atoms via the same mechanism as nanoparticle formation, shown in Fig 1-2.24 This reaction will not directly affect the effectiveness of modified silica nanoparticles, as it attacks silyl groups still baring two or more hydroxyls, but is a concern when unmodified controls are used.4, 24 Additionally, the equilibrium is not significantly in favor of catechol attachment below 90 °C.24
3
Figure 1- 2. Catechol attack of surface silyl groups24
Although direct binding of molecules to the silica crystal is unfavorable, silane chemistry, employing similar chemistry to silica particle formation, is well researched and very robust.20, 27, 28, 32, 37-40 Bottom up synthesis of silica particles via silane monomers leaves most if not all of the surface hydroxyl groups available for further synthesis, thus removing the need to activate the silica surface.24, 41 Typical modification techniques involve addition via silane chemistry or direct modification of surface hydroxyls.4, 21, 22, 32, 42
Research into the surface of silica particles began in 1936 when Kiselev first proposed that the active surface of silica was characterized by hydroxyl groups coating the surface of the SiO2 bulk and in the next two odd decades research into the activity of silyl groups was probed by a number of groups each contributing a different piece of the puzzle.24 Then in 1959, Belyakova et al. published a review showing that the silica active layer is the only layer that participates in silane chemistry or organic molecule chemisorption.24, 29 By summarizing the work of the preceding decade, Belyakova et al. were able to surmise that unless micropores were involved, such as those in controlled pore glass, the SiO2 bulk in a silica particle would not participate in the surrounding chemistry.29 Many groups since then have utilized this principle to embed and protect dyes or other active species inside a silica core.32, 43-46 This finding also means that most of the nanoparticle’s characteristics would be defined by its surface identity, enhancing the importance of surface modification on silica particle behavior.4, 21, 42, 47
4
It is worth noting here that silica nanoparticle formation is a reversible reaction and the silica can indeed dissolve into the surrounding solvent.24 Thus, any surface modification stability is limited by the natural leeching of silica. Fortunately, trace impurities decrease the solubility of silica in water, from 16 to 60 ppm depending on particle size, and ethanol, up to 164 ppm in pure ethanol, and thus surface modifications, acting as trace impurities, increase the stability of the silica nanoparticle.24, 48, 49 Not only is the total equilibrium amount of dissolved silica in solution decreased upon the introduction of impurities and surface modification, but the dissolving rate also decreases with impurities.24 This decrease in dissolution rate is enhanced by a basic environment while being counteracted by the small size and high specific area of nano-sized silica.21, 29 The rate of silica dissolution in a pure solution can be given as:24
= − (1)
where dc/dt is the rate of dissolution, k1 is rate constant of dissolution, k2 is rate constant of deposition, c is the concentration of silica, and S is the specific area of the solid silica.24 Numerous studies using silica nanoparticles as sensors or catalysts have demonstrated stable nanoparticle composition for upwards of 3 months.32, 38, 40, 50, 51 However, the stability of the nanoparticle over time is limited by the silica core stability and by the dye or surface modification group stability.24, 49
Surface modification of silica particles has a long history dating back to around 1968 with the modification of hydrophilic silica surfaces to create hydrophobic surfaces for dispersion of silica in organic solvents.24 Initial efforts into altering silica surfaces mostly involved improving silica dispersion into various media. 52 As shown in Figure 1-3, many of the techniques, such as silane modification22, 32, 52,
53, modification of the silane functional group13, 22, 47, and direct surface modification35, 53-57, have greatly influenced our understanding of silica surface chemistry.
5
Figure 1- 3. Taken from Peng and coworkers, the figure presents the common modifications possible for silica surfaces52
Surface modified nanoparticles continue to be a promising field of research due to the ability to tailor the surface chemistry of the nanoparticles for biochemical sensing and imaging purposes.28, 38, 58-61
Recently, groups have successfully tailored the surface of fluorescent particles to target cancer cells or interesting marker proteins for relevant diseases.47, 62-67 This type of surface chemistry is of particular interest in the work presented as it involves the covalent incorporation of a dye onto the surface o either through thiol or silane chemistry.13, 27, 32, 65, 66 Many of these syntheses feature a silanized dye either on the surface or incorporated into the matrix core of the particle.32, 40, 59, 65 This research thesis focuses on the former in an attempt to synthesize silica nanoparticles with surface bound chromophores and functional groups for further modifications with receptor groups that may affect the photochemistry of the dye..
1.2 Fluorescence Basics and Quenching Theories
When a molecule becomes excited via light absorption, there are a number of possible processes that can take place in its return to the ground state.69, 70
6
Fluorescence is the emission of photon from an excited molecule as it returns to the ground state. The emitted photon is typically of a lower energy due to a large amount of alternative intermediate pathways such as vibrational relaxation, contact energy transfer, or loss as heat shown in Fig 1-4.69, 70, 71 This phenomenon depends upon the difference between the initially populated molecules orbital’s energy levels in a particular fluorescent molecule as a function of environment.69, 70 Research to alter the effect of the environment of the dye molecule and their corresponding spectral changes has been reviewed recently.11, 52, 61, 72-76
Figure 1- 4. Diagram of possible collisional energy loss pathways. (a) Thermal relaxation (b) Energy loss as heat (c) Fluorescence (d) Energy transfer (e) Phosphorescence70, 71
When light of the correct wavelength is absorbed, the electron is excited to a higher energy level, an excited state (GS to ES in Figure 1-4).60, 69, 70 From that excited state, the electron can return to the ground state by losing energy through a variety of pathways based on the electron states of the molecule.69, 70
Fluorescence is when the molecule’s electron loses the energy from its singlet excited state via photon
7 emission.70, 77 The statistical likelihood of photon emission is dependent upon the identity of the molecule based on the possible electron states, i.e. molecular orbitals, and the relative populations of electron states.69 This statistical likelihood is known as quantum yield of a fluorescent molecule and quantitates the efficiency of the fluorescence process.38, 69, 77, 78
# ɸ= (2) #
Alternative relaxation states are possible photon emission.69 During electron relaxation, the fluorescence pathway for energy loss may not be directly available, the electron could then drop down to the nearby more available lower energy state before returning to ground state.69, 70 Both fluorescence transitions and non-fluorescent transitions may not happen instantaneously as the electron needs time to relax back to a lower energy state. The time between excitation and emission is known as the fluorescence lifetime and typically can range from a manner of nanoseconds to half a microsecond.69, 70 With the advent of new instrumentation, it is now possible to probe the fluorescence lifetime and use it as a measure to determine whether the substrate is interacting with the fluorescent source.70 While we do not utilize this method for probing fluorescence interactions here, it is necessary to describe, since as much work is done in this area and many conclusions we draw are based on information obtained via this technique.
Fluorescence dyes have garnered significant interest as a labeling technique and potential analytical probe because of their high theoretical quantum yield, defined as the ratio of photons emitted over photons absorbed.69, 77 Under the right conditions with the right fluorescent chromophore, a single excitation photon is expected to be enough to incite the release of an emission photon thus making it a more sensitive chromophore compared to UV-VIS active chromophores.69, 70 Additionally, the dyes can be tailored to interact with certain molecules and turn-off or turn-on under set conditions.74, 79-82 Multiple systems,13, 32, 47, 51, 52, 83-89 including the work presented here, exploit this characteristic of fluorescent molecule to generate probes that can sense a variety of substrates at sub-nanomolar concentrations.14, 81, 90
8
Besides quantum yield and fluorescence lifetime, the actual emission spectra may also shift under the influence of interacting substrates or local matrix effects.70, 91-93 Both influences can cause a bathochromic or hypochromic shift in the emission peak, as described below, suggesting interaction between the molecule and surrounding environment.69-71
A bathochromic shift, or red shift, is the emission peak shifts to a longer wavelength, or lower energy, whereas a hypochromic shift, or blue shift, is when the emission peak shifts to a shorter wavelength, or higher energy.60, 70, 71 These shifts signify a change in energy for the emitted photon in fluorescence and can be indicative of enhanced solvent molecules interactions or a change in the local matrix,60, 69, 70 such as the surface modified nanoparticles. The possible explanations for these shifts are complex and information is often presented on a per system basis.70, 94-97 A lack of a unifying theory to predict fluorescence energy shifts has led to continued research in the field of fluorescent dye functionalized nanoparticles. One such energy interaction whose mechanism is not fully understood is fluorescent quenching.
Upon the addition of molecule 2 (figure 1-4), excited state electrons in molecules 1 can return to ground state through intermolecular pathways.70, 71 When this pathway becomes competitive with the native fluorescent emission pathway, the molecule will no longer emitt.69-71 In this case, the molecule is said to be quenched.70
Fluorescent molecules can be quenched in a variety of ways, sometimes even the inclusion of a charged salt molecule can decrease the quantum yield of a fluorescent dye.15, 46, 98, 99 Fluorescent molecules sometimes exhibit quenching behavior when in proximity to metal ions,32, 90, 96 conjugated organic molecules6, 100-102, and other dyes13, 75, 103-106. The specific quenching mechanism and its efficiency is unique for each substrate, but can be categorized into two categories: static quenching or Forster resonance energy transfer. Systems when two molecules bind directly are often observed to form a non-
9 emissive complex, while energy transfer pathways tend to decrease the efficiency of the fluorescence through a distance dependent effect.32 Rurack and coworkers recently40 reported the formation of a Cu2+ terpyridine single molecule complex that was able to favor distance based energy transfer over the more common non-emissive complex formation typically seen for Cu2+ complexes or other single molecule metal bound chromophores such as those synthesized by Lu and coworkers 107 and Wang and coworkers
90.
Forster resonance energy transfer (FRET) was first proposed by Theodore Forster in 1948 and refers to the energy transfer between two chromophores.52 When the absorption band of Dye 1 overlaps with the emission band of Dye 2 the emission can be quenched by Dye 2 absorbing the energy released in emission from Dye 1.52, 61, 108, 109 During energy transfer, Dye 2, if fluorescent, could emit at a longer wavelength than the initially excited chromophore. The result is that the system emits from Dye 2 after being excited at Dye1’s absorption wavelength.52, 70 If the energy transfer pathway is then broken, Dye 1’s fluorescence could be recovered.52, 75, 110 This property of the FRET system allows for in vivo probing of biological systems without cell death or analysis of the interactions between systems or domains too small for optical or electric microscopy.52, 61, 62, 111 FRET has been utilized in probing microcellular interactions111, protein and enzyme structure analysis91, 112-116, ratiometric sensing32, 96, 109, 117-119, and solar energy120-127.
10
Figure 1- 5. Diagram for Forster resonance energy transfer13, 32, 52
FRET is affected by energy transfer efficiency, E, between the chromophores and the distance between them52, 61. Their relationship is given below:
= (3) ∑
An alternative equation looks strictly at the distance between the two chromophores given a distance at which quantum efficiency is 50%:
= (4) ( )
The quantum efficiency is found to be related to the distance by 1/(r6)52, due to the dipole-dipole coupling nature of FRET75, 106, meaning that the efficiency of the interaction drops off by a factor of distance to the power of six. This means typical FRET distances are less than 10 nm with exaggerated signal drop off, thus making FRET especially useful for domain distance probing.61, 128, 129 In equation (4), the r0 term is the overlap between the absorption efficiency of the receiving chromophore and the
11 emission spectra of the transmitting chromophore; the larger the overlap between the two the higher the expected efficiency.52, 75
9000 (ln10) = 128
2 Where Q0 is the fluorescence quantum yield of the donor, k is the dipole orientation, J represents the spectral overlap, n is the refractive index of the solution and N is Avogadro’s number. Since the quantum yield of the FRET system is defined as the number of photons emitted by the receiving chromophore, the quantum yield fluorescence in the receiving chromophore must be accounted for as well. Most of the variables in the last two equations are unique to each FRET pair.52, 61
Recent research into FRET quenching has seen a number of nanosensor based FRET probes synthesized as metal ion detection probes with sensitivity in the micromolar range.13, 52, 84, 109 Research presented by Mancin features a ratiometric dye system with FRET potential as metal ion sensors that respond as both a turn-on and turn-off sensor depending on the wavelength of light chosen for fluorescence sensing.13, 32 Their nanoparticles were synthesized with a silica core containing one part of a
FRET pair while the outer surface is decorated with the second dye in the FRET pair and a metal ion binding site.32 Other research into the field of FRET based nanosensors used modified quantum dots.13, 52,
130 The quantum dot luminescence was altered upon the introduction of a target substrate and deactivated the FRET pathway, causing turn-on fluorescence.131
12
Figure 1- 6. Figure reproduced from Peng and coworkers, diagram of nanorod functionalized gold nanoparticle being FRET quenched by quantum dots. With signal return upon the introduction of TNT52
Although the research presented here does not feature the use of FRET with two dyes. FRET quenching for detection of alternative substrates allows for greater customization of the DDSN system to probe into the intricacies of fluorescent molecules and their interactions with the surrounding species and could be applicable to the system probed in this study.
1.3 Terpyridine as a Method Ion Binding Receptor
Research with terpyridine has been primarily focused on its incorporation into polymer chains132-138 and the tuning of terpyridine properties via the metal ions it is bound to136, 139-148. Interest has increased in recent years for terpyridine as a possible photon receiver and electron donator in solar cells due to their customizability in polymer matrices.82, 149-153
Terpyridine is a tridentate chelator capable of binding to most all metal ions typically in a dimeric fashion.153
13
Figure 1- 7. Diagram of metal ion bound terpyridine, presented with interchangable solvent molecules
Although not having a distinctive visible absorption spectra on its own, metal bound terpyridine can take on the intense color due to the metal-ligand charge transfer band (MLCT).127, 153-157 The absorption spectra of the metal-terpyridine complex can be further tuned by adding active groups onto the terpyridine moiety during synthesis or coupling the terpyridine moiety onto conjugated polymer chains.133, 148, 158-162
This versatility led to research into its potential use as a co-polymer active site for polymer solar cells.141,
153
Figure 1- 8. Common modified terpyridine moieties
Research into the customizability of the terpyridine spectra typically utilizes terpyridine as a charge transfer complex that can pass electrons down an attached conjugated polymer backbone chain or solid support.127, 141, 153, 163, 164 The absorption profile of the polymer could then be tailored by simply varying
14 the conditions during metal-terpyridine complex formation.153, 165, 166 Despite being highly customizable, the synthetic cost of such a polymer may make commercialization difficult.146, 153, 162, 164 Thus research has shifted to the self-assembly of terpyridine containing polymers, thus decreasing the cost for synthetic design while ideally retaining terpyridine’s charge transfer ability.146, 153, 164, 165, 167
Other researcher explored terpyridine and its derivatives’ ability to act as metallo-enzymes through its strong binding to most first row transitional metal ions.139, 153, 168, 169 Specifically, strong binding of Ni2+,
Cu2+,Fe2+/3+, and Pt2+ are of particular interest due to the bioavailablity of enzymes containing Ni2+, Cu2+, and Fe2+/3+ and the strong catalytic activity of Pt2+ for a variety of reactions from hydrogenation to ring formation.153, 170 When coupled with aromatic conjugated terpyridine moieties, the terpyridine is believed to be able to transfer electrons to the metal ion center or absorb unwanted electrons into the ground during catalytic activity.82, 153, 171
In addition to binding most first row transition metals153, terpyridine was shown to bind to other larger metal ions.138, 171-173 Binding to these larger metal ions requires heat and purification via crystallization due to the high energy barrier required to wrap the terpyridine molecule around the larger metal ion.156, 174, 175 Once the complex is formed, it is resistant to both heat and light while retaining most of its charge transfer properties.156, 175, 176 Additionally, the complexes tend to be luminescent allowing for the creation of fluorescence terpyrdine complexes with tunable fluorescence profiles.176 This property makes those metal-terpyrdine complexes attractive for single complex sensing since the terpyridine moiety can be modified to contain an additional binding site or tuned for specific absorption-emission wavelengths. Metal ions such as Ru2+ and Ir2+ can even undergo redox reactions while still attached to terpyridine.160, 177
The potential redox reactivity and chromophore properties of the Ru2+ and Ir2+ terpyridine complexes has led to their use as charge transfer complexes as well as catalysts,153 due to the transfer of 2 electrons during the reaction instead of one electron for most standard redox pairs.178 This two electron transfer
15 increases the possibility for artificial photosynthesis to occur because only two redox reaction pairs are required to happen simultaneously in contrast to the four for a single electron transfer pathway.179
2H +e =H (6)
2H O → 4H +O + 4e (7)
When a current is applied, Ru2+ and Ir2+ terpyridine polymers have the potential to catalyze both hydrogen reduction as well as oxygen oxidation in artificial photosynthesis. Metal-terpyridine containing polymer with photosensitizing dyes or electron sinks were reported to increase the electron density in the terpyridine polymer chain increasing the rate of artificial photosynthesis.146, 153, 160
Extending the photosensitive properties of terpyridine-Ru containing polymers, researchers have cross-linked the terpyridine containing polymers with other polymers to increase electrical conductivity or light absorption in metal organic frameworks, MOFs.180 MOFs are multiple polymers cross linked from individual monomers to form a larger web like complex with a large amount of customizability.153 Much like the coupling of metal-terpyridine units to a single polymer chain, construction of MOFs typically uses self-assembling polymer monomers to create a MOF with the desired properties. By varying the ratio of any single polymer or synthesis conditions, a MOF’s properties can be fine-tuned.
Another field of research utilizing terpyridine complexes revolves around their use as luminescent analytical probes.82, 153, 181, 182 By modifying the terpyridine moiety, researchers have created metal ion sensors able to selectively detect a variety of first row transitional metal ions.40, 82, 146
1.4 Metal Ion Sensing
Metal ion sensing in an aqueous environment is important due to widespread metal ion contamination in water sources from industrial contamination or spillage.9 As more and more countries embrace modern industrial practices, the waste generated from mining for precious metals, modern industrial processes,
16 and waste treatment must be monitored and properly disposed.32, 183 Part of ensuring the correct and safe disposal of waste is through the ability to monitor waste spillage and metal ion concentrations in water sources.
Human consumption of contaminated water causes heavy metal build up in the body leading to renal failure or heavy metal poisoning.184 Additionally, excess metal ions in the environment may affect the local fauna and increase the rate at which they build up in the human body through the consumption of said fauna.70, 185 With the potential impact in mind, the ability to sensitively and selectively monitor metal ion concentrations in aqueous systems with a variety of matrix effects with high precision and reproducibility remains an active research field.
Most analytical techniques for sensing metal ion concentrations at low levels typically employ atomic spectrometry which requires the use of high purity gases to generate the plasma or flame required for the spectrometer’s sensitivity.70, 186 This technique is not field work friendly and much research has been devoted into developing cheaper alternative sensing methods with comparable selectivity and sensitivity that can be employed at the site of sample gathering.70
One such field of research is molecular metal ion sensors, much like the well-known reaction between metal ions and ethylenediaminetetraacetic acid (EDTA), the molecular sensors developed could change color after binding with a particular metal ion. However, the sensitivity of colorimetric sensors does not compare with that from atomic spectroscopy.70, 71 Research done by Wang and coworkers describes the synthesis of a fluorescent molecular sensor utilizing a dansyl dye bound bipyridine ligand. The molecular sensor targets Fe3+ ions, which bind to bipyridine and the dansyl signal is quenched. The sensitivity of such a sensor is reported to be in the micromolar range but is restricted to organic solvent systems due to the hydrophobic nature of both the dye and the chelate. 90 Another sensor was designed by Zhang and coworkers as a ratiometric sensor was capable of Fe3+ detection in blood serum. The sensor consisted of an aromatic imidazole chromophore with a ketone tail shown in figure 1-9. Upon the introduction of Fe3+,
17 the metal catalyzed the deprotection of the ketone to form an aldehyde tail. The molecule undergoes internal charge transfer, red shifting its fluorescence as a function of [Fe3+]. 96
Figure 1- 9. Graphic reproduced from Zhang and coworkers. The figure presented a molecular probe capable of ratiometric Fe3+ detection using fluorescence shifts96
Utilizing similar ideas, quantum dots (QD) have been presented as sensors for detection of metal ions.
Sun and coworkers, employed surface modified CdTe nanoparticles for the detection of Cd2+. The QD is covered by surface bound phenanthroline attached the quantum dot’s thioglycolic acid functionalized surface. The phenanthroline binds to the surface of the quantum dots and quenches the green fluorescence of the CdTe quantum dot. Addition of Cd2+ returns the fluorescence. This research demonstrates the ability of quantum dots to act as sensors that directly utilize their own excitation and emission spectra of without the need for an additional chromophore. 187 The utilization of QD’s for metal ion detection has often been hampered by the difficulty and cost of QD synthesis, modification, and stability.28 Other quantum dots have been employed in a variety of schemes to detect cyanide, food toxins, and other quantum dots.13, 28, 130, 188
In order to engineer robust systems capable of visual and stable analysis of metal ion contamination in water, solid support based sensing has been proposed as a reusable sensing scheme. One such system employs silane chemistry to modify glass slides with a chelate and dye that is able to change colors visually in the presence metal ions.153, 189 The solid support system shows potential as a robust reusable sensor but lacks selectivity and sensitivity when compared to other similar systems due to the relative low
18 surface area of the modified glass slide.153, 190, 191 Further research is being conducted to convert these solid support systems into disease related sensors as their robustness and relatively low cost is appealing.89, 192, 193
Falling between solid support and quantum dots in terms of size and surface area are nanoparticles.
Nanoparticles can be dispersed in solvents while being easier to modify, synthesize, and isolate.24, 52
Modified nanoparticles were used as disease targeting systems66, 194, 195, biological target labeling schemes196, 197, and sensors13, 22, 32, 198 due to their customizability, high functional group loading, and ease of dispersion compared to micron sized particles.199, 200 As they can be chemically modified to suit a variety of purposes, gold, silver, iron oxide, and silica nanoparticles have received particular interest in recent years.10, 20, 32, 40, 199, 201-204
Gold nanoparticles were used in sensors due to their easy modification via disulfide bonding to the surface and gold’s inherent inertness.10, 39, 202, 205 Initial efforts featured gold nanoparticles modified with biological target binding groups such as antibodies that bind to cell targets staining the region with different colors based on the size of the gold nanoparticle.15, 202 Drug molecules were attached to the surface of 15 nm gold nanoparticles and were successfully delivered to bacterial and diseased cells in vitro.128 The advantage of this type of delivery system is the ability to target cells or bacteria via surface modification of the nanoparticle increasing drug potency. When enhanced by the large surface area to volume ratio afforded by nanoparticles this can lead to a large increase in the local concentration of the drug without increasing the dosage.15, 19, 128
Silver nanoparticles were similarly explored for their potential disease curing abilities. Of particular note is a dendrimer modified silver nanoparticle that excreted renally from the human body when under
20 nm in diameter.206 Although the ability to target specific cancer cells was not demonstrated, this result demonstrates potential for disease treatments or drug delivery vehicles via silver nanoparticle technology.
19
Iron oxide nanoparticles, primarily Fe2O3, have been developed primarily for sensor and catalytic applications.207-209 Iron oxide nanoparticles, 10-50 nm in diameter, synthesized via oil emulsion followed by surface modification gave particles for MRI imaging agents or sensors.210, 211 Iron oxide differs from silver and gold nanoparticles in that the iron center is high spin resulting in a strong magnetic moment.39,
128, 194, 212 It is this property that allows the iron oxide core to function as targeted imaging agents after
39, 210, 213 surface modification. Recent research also looked into Fe2O3 nanoparticles as magnetic storage devices much like current hard drives.214
In additional to these three types of nanoparticles, silica nanoparticles have also been ised for similar sensing and catalytic applications.12, 15, 215 Silica nanoparticles can be cheaply prepared compared to gold and silver nanoparticles.24, 25, 33, 54 Unlike the other metal based nanoparticles, silica is magnetically and optically inert thus providing a blank canvas for which the behavior of the nanoparticle is almost solely determined by the surface modifications applied.24, 185 Research was conducted for sensing and catalysis similar to chemistry done on glass slides.22, 36 For the research described in this thesis, we focus on the use of silica nanoparticles as metal ion sensors.
Silica nanoparticle based metal ion sensors have been successful in detecting Pb2+, Cu2+, Fe3+, Ni2+, and Co2+ with targeted surface modification.32, 40, 90 Lead ions were successfully detected using thiol modified dansyl dye doped nanoparticles that were able to detect Pb2+ concentrations in the micromolar range in acetonitrile.32 Copper(II) and iron(III) were successfully detected using a terpyridine functionalized rhodamine dye doped silica nanoparticle similarly in acetonitle.40 Nickel(II) was similarly detected through the use of a nitrilotriacetic acid functionalized tetramethyl rhodamine doped nanoparticle system. 216 The surface of the nanoparticle was closely packed with these molecules resulting in a single
Cu2+ or Pb2+ being able to quench upwards of 13 surface-bound dye molecules.40, 41
Each of the systems presented above were able to detect specific metal ions with a great deal of specificity, however those systems required the nanoparticle to be dispersed in an organic solvent40 or lacked in sensitivity216. By using a strong metal chelate and co-condensing amino-propyl silane or other
20 polar modifiers in an optimized ratio, we predict that the surface-optimized nanoparticles will be able to sensitively and selectively detect the desired metal ions while keeping the nanoparticles dispersible in water or alcohol-water mixtures with controlled pH. In this work we will
The dye-doped silica nanoparticles (DDSN) synthesized here utilize a surface tethered 2’ 2:6’ 2- terpyridine (terpy) as the metal binding site due to its high binding constant for most first row transitional metals as shown in table 1.
Table 1. Stability Constants for Metal Bound Terpyrinde153
Mn2+ Fe2+ Co2+ Ni2+ Zn2+ Cd2+
log K1 4.4 7.1 8.4 10.7 6.7 5.1 log K 13.8 9.9 11.1 5.2 1
Terpyridine was extensively studied for its use in metal organic frameworks (MOFs) as well as its potential use in solar cells.132, 153, 154, 162 Additionally, terpyridine exhibits varied fluorescence and absorbance behavior based on metal ion binding.153 Here, we aim to utilize terpy as a means to tailor the response of the nanosensor to different metal ions and to study the quenching effects as a function of the composition of the DDSN surface chemistry. It may even help us further probe Foster Resonance Energy
Transfer (FRET) dye quenching pathways.
1.5 Turn-on Sensors
In a conventional indicator displacement assay (IDA), a metal binding chelate with open coordination sites binds to a specific metal ion to form a metal-chelate complex that can then bind a dye altering the dye’s emission characteristics. A substrate is then added to compete with the dye for the chelate-metal binding site, thereby releasing the dye into the surrounding substrate and coloring the solution.217, 218 A similar scheme was devised for fluorescent dyes, as the fluorescent dye is quenched upon binding to the metal ion and its fluorescence restored upon displacement from the metal ion in organic solutions.218 The
21 dye affinity for the metal ion versus the substrate affinity for the metal ion determine selectivity of the system. Some dyes bind directly to a variety of metal ions but do not offer the ability to tailor the binding or spectroscopic behavior of the dye. By placing the metal ions on a chelate next to the dye rather than have the dye directly interacting with the metal ion, this system potentially opens up more customizability at the surface of the molecule. Coupled with the use of DDSNPs, a fluorescent dye that will be quenched in the proximity of the metal ion-chelate complex allows for testing to be done directly on the suspended nanoparticle without the need for separation.84, 219, 220
By modifying an older IDA concept, Rurack and coworkers proposed a scheme for a turn-on sensor by introducing an anion to compete with the metal chelate whose nanoparticle fluorescence is quenched under the presence of metal ions as shown in Figure 1-10, calling these quencher displacement assays
(QDA).40 By inducing a signal change through the removal of the metal ion, the sensor can now be used to detect anions and other metal binding molecules.128, 218, 221, 222
Figure 1- 10. Figures reproduced from Rurack and coworkers Shows the scheme proposed for the quencher displacement assay using a modified LUDOX silica core with terpyridine groups and rhodamine B surface functionalization.40
22
Figure 1- 11. Figure reproduced from Rurack and coworkers Shows the results of added anions in the quencher displacement assay scheme at 5 equivalents versus metal ion concentration (1 µM)40
.
2+ 2+ 2+ 2+ The rhodamine B functionalized SNP’s fluorescence was quenched by Cu , Fe , Ni , Mn , listed in order of quenching efficiency, with Cu2+ chosen as the target metal-terpyridine system for quencher displacement assays.40 The fluorescent lifetimes for first row transitional metal bound nanoparticles increased with metal ion concentration suggesting a distance based effect between the bound metal ion complex and the dye direct interaction between the dye and the metal ion, known as static quenching or contact quenching. The team of researchers tested a variety of hard to detect anions, including fluoride, chloride, bromide, iodide, nitrate, sulfate, and phosphate, and discovered that phosphate presented the largest return in signal at 1 uM as shown in Figure 1-11. The team suggested that, due to the return in the fluorescent lifetimes of the system upon the introduction of the anion, that the metal ion must have been removed from the terpyridine in order to recover the fluorescence signal.40 This research lays the foundation for a system that could potentially offer sensitive and selective sensing of anions in a variety of solvent conditions.80, 218, 221 By altering the surface chemistry, the metal bound nanoparticles could be used to detect these molecules by modifying the terpyridine or silica nanoparticle surface.
In this work we will present a new terpyridine surface functionalized DDSN, with alterations to surface composition and dye identity which enable it to respond very sensitively to copper ions (see Fig
1-12). We will present data on this system activity as a potential QDA system much like the the system developed by Rurack and coworkers.40 Quencher displacement assays require removal of the quenching
23 species by substrate addition to recover the signal as the primary sensing technique (turn on sensor). We propose that substrate binding to the terpy-Cu chelate may affect energy transfer quenching without displacement of the metal ion.
Figure 1-12 the sensing scheme used in our work for both metal ion binding that turn-off dye emission and the turn-on emission via binding of biologically relevant targets.4, 13, 76, 221, 223 This hypothesis is tested by changing the terpyridine/dye/modifier ratio to study metal ion quenching and subsequent addition of metal binding substrates to restore dye emission to offer some insight into FRET or other type of energy transfer.40
Figure 1- 12. Diagram for our quencher displacement assay surface
24
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217. Comes, M.; Aznar, E.; Moragues, M.; Marcos, M. D.; Martinez-Manez, R.; Sancenon, F.; Soto, J.; Villaescusa, L. A.; Gil, L.; Amoros, P., Mesoporous hybrid materials containing nanoscopic "binding pockets" for colorimetric anion signaling in water by using displacement assays. Chemistry 2009, 15, 9024-33. 218. Wang, K.; Qian, J.; Jiang, D.; Yang, Z.; Du, X.; Wang, K., Onsite naked eye determination of cysteine and homocysteine using quencher displacement-induced fluorescence recovery of the dual- emission hybrid probes with desired intensity ratio. Biosens Bioelectron 2014, 65C, 83-90. 219. Liang, S.; Shephard, K.; Pierce, D. T.; Zhao, J. X., Effects of a nanoscale silica matrix on the fluorescence quantum yield of encapsulated dye molecules. Nanoscale 2013, 5, 9365-73. 220. Casciato, S. L.; Liljestrand, H. M.; Holcombe, J. A., Development of a fluorescence model for the determination of constants associated with binding, quenching, and Forster resonance energy transfer efficiency. Anal Chim Acta 2014, 813, 77-82. 221. Martinez-Manez, R.; Sancenon, F., New advances in fluorogenic anion chemosensors. J Fluoresc 2005, 15, 267-85. 222. You, L.; Zha, D.; Anslyn, E. V., Recent Advances in Supramolecular Analytical Chemistry Using Optical Sensing. Chem Rev 2015. 223. Barba-Bon, A.; Costero, A. M.; Gil, S.; Parra, M.; Soto, J.; Martinez-Manez, R.; Sancenon, F., A new selective fluorogenic probe for trivalent cations. Chem Commun (Camb) 2012, 48, 3000-2.
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Chapter 2: Experimental
2.1 Materials
Ammonium hydroxide (28%wt, ACS reagent grade) was purchased from Mallinckrodt and diluted with deionized water to the desired concentration. The following were purchased from Sigma Aldrtich: 3-
Aminopropyltriethoxysilane (APS, 99.0%), 2’2’;6’2’-ChloroTerpyridine (>98.0%), deuterated chloroform (99.8% deuterated), ethyl acetate (99.9%, CHROMASOLV® Plus grade), tetraethyl orthosilicate (TEOS, 99.0%), toluene (99.9%, CHROMASOLV® Plus grade), 3-(triethoxyslyl)propyl isocyante (95%), KOH (powdered, 99.9%), 5-amino-1-pentanol (95%) acetonitrile (99%,
CHROMASOLV Plus grade), fluorescein thioisocyante (98%), and rhodamine B thioisocyante (>95%).
KOH 100%, pallets) were obtained from Alfa Aesar. Absolute ethanol (200 proof, USP) was purchased from KOPTEC. DI water was obtained with an Aquatec purification system at USF. Lead nitrate (ACS reagent grade) was purchased from EM Science. All reagents were used without further purification unless otherwise stated.
*metal ion salts and their suppliers
2.2 Instrumentation
2.2.1 Centrifugation
Centrifugation was performed using an Eppendorf Centrifuge 5804R in Amicon YM-10 filtration units. The top plastic locking portion of the filter contains two parts, a larger locking unit and a smaller cap at the top. The cap was kept on during movement to avoid spillage but removed prior to filtration.
The synthesized nanoparticle (NP) suspension was transferred equally from the reaction round bottom flask to the plastic non-filter containing portion of two Amicon YM-10 filtration units via a plastic pipette. Ethanol was added to the filtration units until the liquid level met the mark on the filtration unit, approximately 25 mL each. The filter unit was added to the filtration unit’s container and the top locked into place.
The filtration units were placed opposite each other in the Eppendorf Vulcan tube holding rotor making sure the rotor was balanced. The centrifuge top was then closed and an audible locking sound was
37 heard before setting the speed and temperature of the filtration run. The filtration units were spun at 2950 rpm at 20 °C for 40 min. At the end of that time, the filtrate was decanted by removing the filter portion of the unit and dumping the contents into a waste container. Pure ethanol was then added to the concentrated reaction mixture and further centrifugation was carried out with the same filter unit. This process was repeated for at least 5 runs to wash the particles of monomers.
If dye attachment was performed, the end point for filtration was monitored via UV-Vis analysis of the filtrate for dye. If no visible or UV active dye were incorporated, then the filtration was continued until there was no olfactory detection of ammonium hydroxide in the filtrate. Alternatively, the filtrate was added to an equal amount of water and tested for pH. If pH was 9 or lower, the purification was considered complete.
The remaining suspension in the filtration unit was then transferred quantitatively into a 20 mL glass vial. The total washing ethanol used did not exceed 5 times the remaining suspension volume as a very dilute particle suspension will result. The vial was parafilmed and covered with aluminum foil before being stored at 4 °C.
2.2.2 Fluorescence
Fluorescence spectra were obtained on a Horiba Jobin Yvon FluoroMax-4 Spectrophotometer. The particles were suspended in their native solvent via sonication for 30 min followed by dilution into quartz fluorescence cells.
The stored glass vials were taken out of the fridge, warmed to 25 °C, and placed into a beaker filled with water and sonicated for 30 min in a water bath sonicator to re-suspend them in ethanol. After sonication, 5-10 uL of the concentrated suspension were added to a quartz cell containing 2 mL of the desired solvent mixture. The actual volume of nanoparticle suspension added was determined by the desired fluorescence counts and thus each batch of dye doped silica nanoparticles (DDSNP) had slightly different volume added requirements. The target count amount for any DDSNP sample was around 2 million counts as the linear range of the detector was experimentally determined to be approximately
100,000 to 7 million counts.
38
A quartz cell filled with the nano-dispersion was then placed in the spectrometer and the excitation and emission wavelengths were selected based on the nature of the dye in the DDSNP. Additionally, an emission detection range was set at 30-50 nm around the emission maximum of the free dye. The emission wavelength minimum was 15 nm longer than the excitation wavelength.
Measurements were taken in triplicate with at least a 1 min break between spectra. Mixing via pipette during the 1 min breaks gave best results especially on SNP with lower dispersion in the desired solvent.
A blank filled with the solvent used was taken prior to each sample at the designated settings to confirm the cleanliness of the cuvette.
2.2.3 Solvent purity
To check for synthesis solvent purity, 0.5 uL of the solvent was injected onto a Varian 430 GC system with an Agilent DB-624 residual solvent analysis column in direct injection mode using a glass microsyringe. Helium was used as a carrier gas. The temperature program started at 40 °C with a 4.5
°C/min ramp to 80 °C with a 1 min hold then a 25 °C/min ramp to 240 °C with a 5 min hold. The resulting chromatogram was compared to a blank solvent run. The absence of any major peak other than the solvent peak was considered sufficient for purity.
2.2.4 Nuclear magnetic resonance
For small molecule identification in the reaction mixture, NMR spectra (1H) were acquired on a
Varian 500MHz NMR spectrometer. A glass pipette tip of product was transferred into an 5 mm NMR tube with CDCl3 (99%) at around three finger width height. The NMR tube was then capped and inverted several times to ensure that the product was fully dissolved. If the product did not dissolve in the chosen
NMR solvent, D2O or deuterated-DMSO solvent was used.
2.2.5 UV-Vis spectra.
UV-Vis spectra were obtained on a Varian UV-Vis-NIR Spectrophotometer 5000 equipped with a thermostated cell holder, 1 cm quartz cell. Typically, the solution to be analyzed was pipetted into a quartz UV cell containing 2.5-3 mL of solvent. The absorbance spectra were obtained from 200-800 nm
39 via the software. The thermostated cell holder was not utilized in general procedures unless otherwise stated.
2.3 Preparation of 1-(5-([2,2’:6’,2’’-terpyrdin]-4’-yloxy)pentyl)-3-(3-(ethoxylsilyl)propyl)urea
(Terpyridine Silane)
2.3.1 5-([2,2’:6’,2’’-terpyridin]-4’-yloxy)penta-1-amine (amino-terpy)
The method of Shubert and coworkers was modified as below.1 Powdered KOH (3.5 g) was dried in a
3-necked round bottom flask at 105°C in an oil bath under direct vacuum for 48 h to remove trapped water in the KOH. After cooling to 60 °C under nitrogen atmosphere 30 mL of DMSO was added slowly via syringe to dissolve the base, and 5-amino-1-pentanol (crystal, 0.77 g) was added quickly via paper funnel to give a yellow hue. The mixture was stirred under nitrogen at 60 °C for 30 min before 4′-chloro-
2, 2′, 6′, 2″-terpyridine (2.0 g) was added all at once which gave a wine red solution. The mixture was stirred at 60 °C under nitrogen for 48 h and turned yellow. After cooling to room temperature, the reaction mixture was quickly poured into 600 mL of DI water into a beaker using a funnel to form an off white precipitate. After 30 min the product was collected on a Bruker funnel using 30 um paper filters via filtration with an aspirator. The resulting off-white powder was dried overnight under vacuum to yield 2.6 g of product which was used without further purification. NMR characterization was consistent with
2-5 1 literature values and indicated high purity (>99%, 2.5g) H NMR (500MHz, CDCl3): δ = 1.56 [m, 4H,
CH2]; 1.88 [m, 2H, CH2]; 2.76 [t, 2H, CH2]; 4.24 [t, 2H, CH2]; 8.00 [s. 2H, AR-H]; 8.60 [q, 2H, AR-H];
8.67 [q, 2H, AR-H].
2.3.2 Terpyridine Silane
The solid product from above (2.0 g) was added to a bottom flask fitted with a condenser containing
30 ml of dry dichloromethane with mixing to which was added 3-(triethoxysilyl)propyl isocyante (510
μl) via digital pipette. The reaction mixture was refluxed under nitrogen for 48 h. The dichloromethane was removed rotary evaporation after transferring the reaction mixture to a single necked 100 mL round bottom flask and the resulting precipitate was transferred to glass test tubes and washed with n-hexane (20 ml) 3 times. The wash was performed by introducing the hexane mixing to create a suspension,
40 centrifuging the suspension in a bench top centrifuge, then decanting the hexane. Repeated washing yielded an off-white precipitate that was dried in vacuo overnight. The procedure yielded 1.78g of
3 1 product. The product was used without further purification. H NMR (500MHz, CDCl3): δ = 0.56 [q,
2H, Si-CH2]; 1.11-1.16 [m, 13H, CH2 and CH3]; 1.80 [t, 2H, CH2]; 3.15-3.10 [m, 4H, CH2]; 3.64 [q, 2H,
CH2]; 3.71 [q, 6H. O-CH2-Si]; 4.15 [t, 2H, CH2]; 4.41 [s, 1H, NH]; 4.54 [s, 1H, NH]; 7.25 [m, 2H, Ar-H];
7.76 [m, 2H, Ar-H]; 7.92 [s, 2H, Ar-H]; 8.54 [q, 2H, Ar-H]; 8.60 [q, 2H, Ar-H]
2.4 Synthesis of DDSN with terpy and amine surfaces
2.4.1 Two Step Modification of SNP using Fluorescene Isothiocyante (FITC) to Synthesize
Silica Core Terpyridine Amino Dye Shell Silica Nanoparticles (TAD-SNP1)
Our synthetic methodology was modified from the procedure used by Mancin and coworkers 2, 6 .
Tetraethyloxyorthosilane (TEOS, 200 uL) and 0.9 mL of 22% ammonium hydroxide were added to 20 mL of anhydrous ethanol in a 100 ml round bottom flask with stirring for 4 h at room temperature under nitrogen before another portion of 200 uL TEOS and 0.9 mL of 22% ammonium hydroxide were added with another 4 h of stirring under nitrogen. No visible change was observed. Aminopropylsilane (0.02 mmol; 4.6 uL), TEOS (0.25 mmol; 56 uL), and the product above, terpy-silane (0.1 mmol; 50 mg) were then added together and stirred at room temperature under nitrogen for 16 h. The resulting solution was transferred to a plastic syringe fitted with a 0.45 µm membrane filter to remove excessively large particles then concentrated in an Amicon YM-10 unit. Purification was performed via addition of ethanol, and re- concentration of the mixture via centrifuge (repeated 7 times). The resulting suspension was sonicated for
30 min and then passed through another 0.45 µm membrane filter before transferring into a scintillation vial for storage. The nanoparticle solution appeared off-white yellow.
Half of the solution above was transferred into a 50 ml round bottom flask containing 10 mL of ethanol. To this mixture, FITC (6.5 mg, 0.0185 mmol) was added and the reaction stirred under nitrogen at room temperature for 48 h. The solution was passed through a 0.45 um membrane filter as described previously and concentrated on a centrifuge using an Amicon YM-10. Ethanol was added to the resulting
41 retentate and the mixture concentrated again. This process was repeated 9 times until no fluorescein remained in the filtrate as evidenced by the absence of fluorescence upon luminescence of “black light” at
254 nm via a handheld light source. The resulting mixture was sonicated and transferred to a scintillation vial for storage.
2.4.2 One Pot Synthesis of Uniform Core Terpyridine Amino Dye Silica Nanoparticles
(TAD-SNP3)
FITC was allowed to react with neat aminopropyl silane (1:2) under nitrogen atmosphere for 16 h in
25 mL round bottom flask, the resulting bright orange liquid (FITC-silane) was used directly without purification.
Tetraethyloxyorthosilane (TEOS, 200 ul) and 0.9 ml of 22% ammonium hydroxide were added to 20 ml of anhydrous ethanol in a 100 ml round bottom flask with a magnetic stir bar. The reaction was allowed to proceed for 4 hours at room temperature under nitrogen atmosphere during which the reaction solution turned a translucent white. Terpy-silane (67.89 mg) and FITC-silane / aminopropylsilane mixture
(100 ul, 0.01 mmol fluorescein / 0.002 mmol aminopropylsilane) were added to the solution mixture and the reaction was stirred under nitrogen for 16 h. A glass fitted condenser was attached and the reaction was refluxed for an additional 2 h before. The mixture was passed through a 0.45 µm membrane filter, concentrated on an Amicon YM-10 filtration unit to which ethanol was added and the mixture concentrated again. The filtrate was monitored by UV-Vis for the presence of FITC-silane, λmax = 325 nm, until none was detected. The resulting mixture was then sonicated in a water bath for 15 min before transferring to a scintillation vial as a suspension for storage.
2.5 Nanoparticle Concentration
A SNP or DDSNP suspension (1.00 ml) was added to a dried, pre-weighed 20 mL scintillation vial and placed into an oven at 120°C for 1hr. The vial was then allowed to cool before weighing the remaining solid. The mass concentration of the suspension was calculated using the following equation:
( )= (2)
42
2.6 Determination of FITC Loading (mol/mg) on TAD-SNP
FITC (4.59 mg) was dissolved in 10 ml of 1:1 acetonitrile: ethanol with mixing then diluted 100 fold using the same solvent mix. This stock solution was used to make dilutions and measurements of fluorescence intensity versus [FITC] to establish a calibration curve on the fluorimeter with a typical range of 50 nM to 10 uM [FITC]. Fluorescence readings on nanoparticle solutions (typically 0.04 mg/ml solutions) were compared to the calibration curve to establish dye loading. (λex = 500 nm ; λem = 525 nm)
[ ] = ∗ [ ]
2.7 Determination of TAD-SNP Solubility and Stability
A TAD-SNP solution (0.5 mg/ml) was prepared in the desired solvent mixture. The solution absorption and fluorescence spectra were recorded over the course of an hour to determine changes in intensity. (λex = 500 nm ; λem = 525 nm)
2.8 Metal Ion Quenching Assay (Turn-off Sensing)2
Metal nitrate stock solutions (1.0 mM) in DI water were prepared via gravimetric addition of the dry salt into volumetric flasks followed by the appropriate dilution. Aliquots of less than 50 µl were added to
3.00 ml of 20 µg/ml DDSNP solutions, prepared by addition of DDSNP with known concentration into
3mL of 1:1 acetonitrile: ethanol using a digital pipette, and allowed to equilibrate for 30 min before fluorescence measurements were recorded in triplicate. (λex = 500 nm ; λem = 525 nm)
2.9 Quencher Disruption Assay(Turn-on Sensing)3
Eight microliters of 1.0 mM metal nitrate solution were added to 3.00 ml of 20 ug/ml solution of
DDSNP in 1:1 acetonitrile: ethanol and allowed to equilibrate for 30 min. This quenched fluorescence served as a baseline starting point. Small aliquots, up to 100 ul, of the desired substrate in DI water (10 mM) were added to the metal-TAD-SNP solution and after waiting for signal stability (30 min), the fluorescence was recorded in triplicate.
43
Work Cited
1. Schubert, U. S.; Alexeev, A.; Andres, P. R., Terpyridine-Functionalized TentaGel Microbeads: Synthesis, Metal Chelation and First Sequential Complexation. Macromolecular Materials and Engineering 2003, 288, 852-860. 2. Arduini, M.; Mancin, F.; Tecilla, P.; Tonellato, U., Self-organized fluorescent nanosensors for ratiometric Pb2+ detection. Langmuir 2007, 23, 8632-6. 3. Calero, P.; Hecht, M.; Martinez-Manez, R.; Sancenon, F.; Soto, J.; Vivancos, J. L.; Rurack, K., Silica nanoparticles functionalised with cation coordination sites and fluorophores for the differential sensing of anions in a quencher displacement assay (QDA). Chem Commun (Camb) 2011, 47, 10599-601. 4. Schubert, U. S., Terpyridine-based Materials: For Catalytic, Optoelectronic and Life Science Applications. Wiley-VCH: 2011; Vol. 3, p 52. 5. Winter, A.; Hoeppener, S.; Newkome, G. R.; Schubert, U. S., Terpyridine-functionalized surfaces: redox-active, switchable, and electroactive nanoarchitecturesgland. Adv Mater 2011, 23, 3484- 98. 6. Bonacchi, S.; Genovese, D.; Juris, R.; Montalti, M.; Prodi, L.; Rampazzo, E.; Sgarzi, M.; Zaccheroni, N., Luminescent chemosensors based on silica nanoparticles. Top Curr Chem 2011, 300, 93- 138.
44
3. Fluorescence Quenching via Metal Ion Binding to TAD-SNP
3.1 Introduction
Recent developments in understanding fluorescence quenching, in which NPs incorporate chromophores, revolve around the measurement and changes in fluorescent lifetimes and signal under the influence of added substrates.1-6 Modern theory suggests two possible types of quenching, static quenching or energy transfer quenching.2, 4, 7-9 Research into the quenching of chromospheres on nanoparticle systems suggests that the specific quenching mechanism may be unique to the system in question.9, 10 For example, binding of large metal ions such as Pb2+ were just as effective at turning off fluorescence of a dansyl based core-shell SNP11, 12 via static quenching compared to the energy transfer quenching observed through the formation of colored complexes between Fe3+ or Cu2+ and surface bound terpyridine.13 A similar effect was reported for Fe3+ and fluorescein dye combinations in both molecular and SNP systems.14 Other groups reported fluorescence quenching of sulforhodamine in the presence of
Fe3+, Cu2+, and Ni2+ via a colored complex,1 and Ru2+ terpyridine polymers were quenched by Zn2+ binding to free terpyridine sites despite not forming a colored complex.15 These results implied that an energy transfer mechanism is a requirement for energy transfer to occur in terpyridine-metal complexes in close proximity to the dye where their absorption spectra overlap with the fluorescent emission.1-15
However, terpyridine bound Pb2+ was able to quench the fluorescence of the dansyl chromophore with no visible spectra overlap. We will show here that Pb2+ can also quench the fluorescence of fluorescein and rhodamine, if a sulfur bound chelator is present on SNP.11 A possible explanation for this was provided by Tonellato, Pb2+ was a larger metal ion and that its electron cloud interacted with the dye-excited state, providing the dye excited state with an alternative energy loss pathway.11, 16 Fluorescence lifetime measurements of Pb2+ in this system suggested that the metal ion interacted with the chromophore but the exact mechanism of interaction was not clear.11 We hope to examine new TAD-SNP systems in order to contribute to an understanding of the quenching mechanisms observed in DDSNP by probing other unexplored potential metal and dye combinations.
45
As others have reported, addition of metal ions to surface bound chelates on DDSNP quenches the dye fluorescence as a function of concentration.17-19 As more metal ions are titrated into the NP suspension, the equilibrium shifts toward bound metal ions via Le Chatelier’s Principle and I0/I
20 increases. The rate at which I0/I increases is dependent on the equilibrium constant between free surface terpyridine and metal bound terpyridine, the binding constant of terpyridine to the metal, and the efficiency of the energy transfer between the metal bound complex and the dye. These relationships are presented as the Stern-Volmer equation21-24:
=1+ [ ] (1)
Where I0 is the initial fluorescence intensity, I is the fluorescence intensity at [Q], Ksv is the quencher
23, rate coefficient or Stern-Volmer constant, τ0 is fluorescence lifetime, and [Q] is quencher concentration.
24 The Stern-Volmer constant includes the metal binding equilibrium constant, energy transfer efficiency, and metal ion diffusion rate.22, 23 When a single interaction is believed to inhibit a photochemical or photophysical process this equation is used. Here, the metal ion provides an alternative energy transfer pathway instead of dye photon emission.
When plotted as I0/I versus [Q], a linear relationship suggests that the fluorescence quenching is caused by an interaction between the nanoparticle based dye and a single metal ion quencher over a given
24 concentration range. As the concentration increases, a continued linear increase of the I0/I ratio cannot be always be observed because of increasing interaction between the metal ions and saturation of all available reaction sites.10, 24-27
In the work described in this thesis, the fluorescence of a SNP bound dye decreases as ambient metal ion concentration increases, and such quenching studies will be compared across four related systems.
The systems are labeled according to their surface identity. A dansyl-terpyridine system (TD-SNP), a fluorescein-terpyridine-amino system (TAF-SNP), and two rhodamine-terpyridine-amino systems
(TxAyRz-SNP), with varied terpyridine:amine ratios, are presented here. Schemes below:
46
Figure 3- 1 Schematic representations of DDSNP surfaces for TD-SNP, TAF-SNP, and TyAzRx-SNP 3.2 Characterization of DDSNP 3.2.1 Dye Loading on DDSNP The DDSNPs synthesized as described in Chapter 2 of this thesis were analyzed for their dye loading. Dye loading is determined by correlating the free dye fluorescence signal intensity of a known concentration in ethanol to the signal of a known mass concentration of DDSN in ethanol (details in Chapter 2). The loading of dye on each SNP is tabulated in Table 3-1. The dye-loading units were normalized to mg dispersion per mL solution of DDSN. Table 3- 1. Nanoparticle and dye concentration. Values determined from dye loading assay and gravimetric nanoparticle analysis post drying of nanoparticle solution. TAF-SNP TAR-SNP Conc. of NP (mg/mL) 4.34 12.43 Dye Concentration (nmol/mL) 2.0 1.6
Mancin and coworkers, whose synthetic technique was the basis for our synthesis, reported similar dye loadings as the fluorescence signal intensity matches that reported at 20 µg/mL DDSNP concentration, approximately 2 million counts.
3.2.2 Visual Terpyridine Test for SNP Dispersions
A pink-purple complex formed upon the addition of Fe(NO3)3(aq) solution to terpyridine functionalized
SNP. A control experiment between free chloro-terpyridine and Fe(NO3)3(aq) solution in DMSO presented similarly. An easily visible color appeared on the SNP after precipitation due to a high local concentration of the complex on the particles. On nanoparticles modified by amine groups alone, the Fe3+ ions probably
47 bind weakly and an off-white precipitate is formed. This test was performed on nanoparticles prior to dye incorporation as qualitative evidence for the successful surface attachment of terpyridine since the pink- purple color of the rhodamine dye (RITC) and yellow-orange color of the fluorescein dye (FITC) interfered with the pink purple color the test relies on.
3.2.3 UV-Vis Characterization of DDSNP The filtrate solution was monitored via UV-Vis for each nanoparticle preparation to determine if unreacted dye monomers were still present. High concentrations of nanoparticles scattered UV-Vis light and gave a large background signal at concentrations high enough for significant dye signals (0.2mg/mL), especially at wavelengths shorter than 600 nm. Thus, UV-Vis quantification was not useful.
3.2.4 Fluorescence Spectra of Dyes on DDSNP The fluorescence spectrum for each dye gave a different peak emission profile but was generally a single broad peak. There was very little fine detail in the fluorescence spectra and the intensity was used to confirm the presence of the dye and determine dye loading through an external calibration curve as described in chapter 2. There was no observed difference between DDSNP and TAD-SNP emission spectra.
3.2.5 Transmission Electron Microscopy Images of DDSNP
The nanoparticles were imaged at the University of California: Berkeley’s Electron Microscope laboratory. A copper wire mesh was immersed in the nanoparticle suspension, approximately 4 mg/mL, and left to dry at room temperature prior to analysis.
48
The images in figure 3-2 and 3-3 (check numbering throughout) suggest that the nanoparticles synthesized are roughly uniform size, 60 nm in diameter, and are mostly spherical. The agglomeration in figure 3-3 is primarily due to the high concentration of the nanoparticles on the copper wire grid post evaporation. This result is consistent with other surface functionalized DDSNPs reported in literature.1, 11, 28-30 At regions of higher DDSN concentration, agglomeration is evident but each distinct sphere is still approximately 60 nm in diameter. The formation of an agglomerated product is a concern but it is unclear whether such agglomerations would form in dilute solutions. As such, sonication was required. The nanoparticles were always sonicated for 30 minutes before use and nanoparticles diluted to a final concentration of 20 µg/mL to minimize agglomeration.
(scale unit: 50 nm)
Figure 3- 2. TEM Photos of FITC-Terpy-NP1 from UC-Berkeley Electron Microscope Lab
49
(scale unit: 0.2 µm)
Figure 3- 3. TEM Photos of FITC-Terpy-NP1 from UC-Berkeley Electron Microscope Lab
3.3 Effect of Added Metal Ions on Various DDSNP Surfaces
3.3.1 Pb2+ with Thiol-coated Dansyl DDSNPs
The synthesis of propyl thiol coated dansyl DDSNP for the detection of Pb2+ followed the protocol laid out by Mancin and coworkers. Adding Pb2+ quenched the fluorescence of the thiol coated DDSNP to approximately 40% original signal, similar to the values reported by Mancin and coworkers. To improve selectivity for transitional metal ions we attached the strong chelator, terpyridine, to the SNP structure using terpy-silane, described in Chap 2.11
3.4 Effect of Metal Ions on Dansyl Dye Immobilized on Terpyridine Functionalized SNP
The DDSNP dispersion synthesized from procedures laid out in chapter 2 was pipetted into acetonitrile to give a final concentration of 20 µg/mL. A fluorescence measurements taken for this dispersion at λex= 297 nm, λem = 500 nm were used as I0. To the DDSNP dispersion, small aliquots of metal ion solution were added. The fluorescence of the system was recorded after pipette mixing and a wait time of 1 min.
50
hv1 hv1 M2+
hv2
M2+ NP +M2+ NP
M2+
Figure 3- 4. Scheme of Fluorescence Quenching upon Introduction of Metal Ions
n+ Table 3-2. % Quenching of initial intensity, I0/I, @ 320 nm TD-SNP and 300 µM M Zn2+ Pb2+ Cu2+ Ni2+ Fe3+ 11.48 20.6 52.63 23.1 63.22
Note: 1:2 EtOH:ACN, 1 mM HEPES Buffer @ pH 7.0; 300 µM metal ion concentration; 80 µg/ml DDSN concentration; where I0 is the original fluorescence intensity and I is the fluorescence intensity after metal ion addition. I0/I is large where the quenching is effective. The effective quenching order was Fe3+ > Cu2+ > Ni2+ ≈ Pb2+ > Zn2+. This result was not surprising due to terpyridine’s greater affinity towards Cu2+ and Fe3+ relative to the other metal ions.31
Table 3-3. Stability Constants for Metal Bound Terpyridine31 Mn2+ Fe2+ Co2+ Ni2+ Zn2+ Cd2+
log K1 4.4 7.1 8.4 10.7 6.7 5.1 log K 13.8 9.9 11.1 5.2 1
2+ 2+ Interestingly, Ni quenched the dansyl emission by the same amount as Pb , which was the target of the thiol-based dansyl nanoparticles synthesized by Mancin and coworkers. The similarity in Ni2+ and Pb2+ quenching in our system may imply that a different quenching mechanism is occurring for terpyridine- metal quenching and the thiol-metal quenching, as metal complex stability does not explain the above results. The electronic properties of Ni2+ and Pb2+ are different due to their size and valence shell electron distribution. Another explanation of quenching effectiveness variation is for M2+ to bind to the surface primary amines provides two total possible metal binding sites for which Ni2+ and Pb2+ may have
51 differing affinities. However, a control performed with dansyl doped SNP with surface amino groups as
3+ 2+ the only possible chelating sites with added Fe and Pb , both gave I0/I of less than 1.1, which is less than 10% quenching.
If the fluorescence quenching mechanism for the first row metal ions were not by electron transfer, then the enhanced quenching abilities for Cu2+ and Fe3+ could be reconciled, as they formed intensely colored complexes when bound to terpyridine.31-33 The absorbance of such intensely colored complexes could overlap with the emission spectra of dansyl dye, leading to FRET, as explained in Chapter 1.
If the zinc ion results are treated as a non-quenching baseline due to dilution effects, then Pb2+ and
Ni2+ are shown to exhibit some quenching behavior at 300 uM (see Tabel 3.1) in contrast to Pb2+ for the thiol based SNP system that quenches to 45% at 300 uM. Pb2+ does not form a colored complex with terpyridine while Ni2+ forms a lightly color pale green complex.31 The quenching mechanism exhibited by
Ni2+ is not clear, but the quenching behavior exhibited by Pb2+ is probably static quenching as observed for thiol-functionalized dansyl DDSNs that exhibited no response to either Fe3+ or Ni2+. Static quenching, or contact quenching, is when the quenching species forms a complex in the ground state prior to excitation causing quenching of the previously fluorescent molecule.
To summarize, the system designed with terpyridine-functionalized dansyl DDSNP exhibited selective quenching when tested against a series of added metal ions. The modified nanoparticles show significant quenching with added Cu2+ and Fe3+ ions while it was not clear why they demonstrated less quenching with Pb2+ and Ni2+. Inspired by the work of Mancin and coworkers and the work of others in
FRET quenching of rhodamine and coumarin dyes, we designed a system to test the effect of different metal ion quenching schemes.
A common fluorescein based activated dye is FITC, which is fluorescein with an activated NCS terminal as shown in Fig 3-5. After reaction with APS and silica nanoparticle surface attachment, the resulting nanoparticles exhibits λex = 497 nm, λem = 520 nm in ethanol solution.
52
Figure 3- 5 Fluorescein propyl triethoxysilane; λex: 490 nm; λem: 525 nm in Ethanol
3.5 Synthesis and Characterization of DDSMP with Surface Terpyridine
APS was co-condensed with the other silane monomers during DDSNP formation in an attempt to improve DDSN dispersability in aqueous solvents by decorating the surface of DDSNP with primary amine groups. The use of an alternative dye and introduction of APTES created a modified DDSNP that was dispersible in an ethanol and acetonitrile mixture much like that of dansyl-terpy-NP. It was stable in solution over long periods and exhibit stable fluorescence in 100% ethanol. The following sections contain the metal quenching results.
3.5.1 Effect of adding M2+/3+ on FAT-NP1 (FITC-TerpyNP)
Fluorescein-silane, APTES, terpyridine-silane, and TEOS were co-condensed to synthesize FAT-
NP1 as per steps laid out in chapter 2. After purification, metal ions were introduced to a 0.5 mg/mL suspension of NP1 in a mixture of 1:1 ethanol:acetonitrile. Figure 3-6 presents the results of a titration between FITC-TerpyNP and Fe3+.
53
3.50E+06 FITC-TerpyNP
3.00E+06 FITC-TerpyNP + 2.0 µM Fe3+ FITC-TerpyNP + 3.0 µM Fe3+ 2.50E+06 FITC-TerpyNP + 4.0 µM Fe3+ 2.00E+06
1.50E+06
1.00E+06 Fluorescence (A.U.) Fluorescence
5.00E+05
0.00E+00 515 520 525 530 535 540 545 550 Wavelength (nm)
Figure 3- 6 Overlay plot of FITC-terpyridine-NP1 (TAF-NP) fluorescence quenching in the presence of increasing amounts of Fe3+ (1:1 ethanol:ACN; 80µg/ml nanoparticle)
3+ The emission peak wavelength, λmax, of the nanoparticle does not shift significantly during Fe addition, but drops in intensity. This means that the addition of Fe3+ does not change the emission profile of the dye, suggesting that Fe3+ does not interact directly but rather forms a complex with terpyridine that allows for non-emissive relaxation. The large changes in the fluorescence for small differences in [Fe3+] indicate that NP1 has a high sensitivity for Fe3+.
2+ Presented below, in Fig 3-7 and 3-8 are the results for a similar quenching experiment for Cu and
Fe2+ over the concentration range of 0.4 µM to 2.0 µM Cu2+.
54
3.50E+06 FITC-TerpyNP FITC-TerpyNP + 0.4 µM Cu2+ 3.00E+06 FITC-TerpyNP + 0.8 µM Cu2+ 2.50E+06 FITC-TerpyNP + 2.0 µM Cu2+
2.00E+06
1.50E+06
1.00E+06
5.00E+05 Fluorescence Intensity (A.U.) Intensity Fluorescence 0.00E+00 515 520 525 530 535 540 545 550 Wavelength (nm)
Figure 3- 7 Overlay plot of FITC-terpyridine-NP1 (TAF-NP) fluorescence quenching in the presence of Cu2+ (1:1 ethanol:ACN; 80µg/ml nanoparticle)
3.00E+06 FITC-TerpyNP 2.50E+06 FITC-TerpyNP + 0.4 µM Fe2+ FITC-TerpyNP + 0.8 µM Fe2+ 2.00E+06 FITC-TerpyNP + 2.0 µM Fe2+
1.50E+06
1.00E+06
Fluorescence (A.U.) Fluorescence 5.00E+05
0.00E+00 515 520 525 530 535 540 545 550 Wavelength
Figure 3- 8 Overlay plot of FITC-terpyridine-NP1 (TAF-NP) fluorescence quenching in the presence of Fe2+ (1:1 ethanol:ACN; 80µg/ml nanoparticle)
55
2.50E+06
2.00E+06
1.50E+06 FITC-TerpyNP
1.00E+06 FITC-TerpyNP + 2.0 µM Zn2+ FITC-TerpyNP + 4.0 µM Zn2+ 5.00E+05 FITC-TerpyNP + 8.0 µM Zn2+ Fluorescence Intensity (A.U.) Intensity Fluorescence FITC-TerpyNP + 16 µM Zn2+ 0.00E+00 515 520 525 530 535 540 545 550 Wavelength (nm)
Figure 3- 9 Quenching of nanoparticle, FITC-terpyridine-NP1, fluorescence under the presence of Zn2+, spectral overlap (solvent: 1:1 ethanol:ACN; 80µg/ml nanoparticle concentration)
The four results combined suggest that quenching of Fe2+ mirrors that of Cu2+ and Fe3+ as the fluorescence decreased dramatically as metal ion concentration increased, in contrast to Figure 3-8 with
Zn2+ for the same quenching conditions.
At 2.0 µM, Zn2+ shows a reduced signal of 20% over the course of the entire experiment but rebounds after additional Zn2+ addition (note Fig 3-9, blue line). In contrast to the quenched fluorescence trend seen for Cu2+, Fe2+/3+ presented in fig 3-6 and 3-7, the results of Zn2+ addition fluorescence are inconclusive and against all other trends observed. This result suggests that Zn2+ may interact with the DDSNP in unexpected ways and thus is not a suitable control or model system.
A better control experiment, addition of a NaCl solution, gave less than 5% lower fluorescence signal at 80 µM, confirming that the metal ions tested are indeed quenching DDSN with little ionic strength effects.
A closer look at quenching, defined as I/I0, at λem maximum of the various metal ions provides insight into the selectivity of this DDSN system. Fig 3-10 (check all figure numbers) summarizes the effect of added metal ions on the emission of FITC-DDSN over the narrow range tested.
56
1 0.9 0.8
0.7 Zn2+ 0.6 Co2+
I/I 0.5 Ni2+ 0.4 Fe3+ 0.3 Cu2+ Pb2+ 0.2 Fe2+ 0.1 0 0 0.5 1 1.5 2 2.5 3 3.5 4 [Mx+]; µM
Figure 3- 10 FITC-Terpy-NP1 quenching sensitivity versus metal ions at a range of concentrations. The nitrate salts were used for all of the metal ions except Fe3+ where the chloride salt was used. (20 µg/ml DDSNP solution; 1:1 Ethanol:ACN)
The results reveal that Zn2+ affects the fluorescence of FITC-Terpy-NP1 the least while Cu2+ and Co2+ are the most effective quenchers. The ranked efficiency of quenching, at 3.4 µM would be: Cu2+ > Co2+ >
Pb2+ > Ni2+ > Fe2+ > Fe3+ >> Zn2+. At 3.4 µM Cu2+, the signal is greater than 90% quenched, so introducing higher concentrations of [Cu2+] will no longer linearly decrease the fluorescence signal.
Reformatting the above data for use in Stern-Volmer plots provides additional information.
=1+ [ ]
Where I0 is the initial fluorescence intensity, I is the fluorescence intensity at [Q], Ksv is the quencher rate coefficient or Stern-Volmer constant, τ0 is fluorescence lifetime without Q, and [Q] is quencher concentration.23, 24 This manipulation allows us to look into the relationship between the metal ion and the
FITC-Terpy-NP1 DDSN. Here the specific τ0 has not been measured and all collected Stern-Volmer constants are presented as relative slopes. Figure 3-11 depicts the data and Stern-Volmer linear fits for non-standard constructs.
57
4 y = 0.7365x + 1.215 3.5 Zn2+ Ni2+ y = 0.6172x + 0.844 3 Fe3+ Pb2+ 2.5 /I 0 I 2 y = 0.3876x + 0.906 1.5
1 y = 0.0885x + 1.0466
0.5 0 0.5 1 1.5 2 2.5 3 3.5 [Mx+] ; uM
Figure 3- 11 Stern-Volmer plot of FITC-Terpy-NP1 versus metal concentration for [Zn2+], [Ni2+],[ Fe3+], and [Pb2+]; (20 µg/mL DDSN solution in 1:1 Ethanol:ACN)
10.5 10 y = 2.6448x + 0.4271 9.5 Zn2+ 9 8.5 8 Co2+ 7.5 7 6.5 Cu2+ 6
/I 5.5 0
I 5 4.5 4 y = 1.7618x + 0.3087 3.5 3 2.5 2 y = 0.0885x + 1.0466 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5 [Mx+] ; uM
Figure 3- 12 Stern-Volmer plot of FITC-Terpy-NP1 versus metal concentration for [Zn2+], [Co2+], and [Cu2+]; (20 µg/mL DDSN solution in 1:1 Ethanol:ACN)
From fig 3-12 we see that while the R2 > 0.9 for Cu2+ and Co2+, the linear fits for these two metals are not a good fit to the linear Stern-Volmer equation as their intercept falls out of the 1±0.3 range. Thus, we fit the data to a polynomial function as in fig 3-13:
58
10.5 10 2 9.5 Zn2+ y = -0.0211x + 0.1585x + 1.0156 9 R² = 0.9784 8.5 8 7.5 y = 0.4458x2 + 0.2807x + 0.9648 7 Co2+ 6.5 R² = 0.9912 6 2 /I 5.5 y = 0.4672x + 1.0924x + 1.1149 0 I 5 Cu2+ 4.5 R² = 0.9941 4 3.5 3 2.5 2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5 [Mx+] ; uM
Figure 3- 13 Polynomial Fit for Stern-Volmer plot of FITC-Terpy-NP1 versus metal concentration for [Cu2+] and [Co2+]
From fig 3-11 and 3-12, the best linear fits of R2 > 0.95 were for Cu2+, Pb2+, and Ni2+. If the data gave a linear fit to the Stern-Volmer equation, the quenching was deemed diffusion-based single substrate
34 response pathways or a “sphere of influence” pathway. The slope, KSV or the Stern-Volmer constant is a measure of the system’s energy transfer effectiveness. Additionally, a high R2 value means that one may quantitatively determine the metal ion concentration over the tested concentration range. The data for
Cu2+, Pb2+, and Ni2+ gave R2 values greater than 0.95, suggesting that this system is indeed capable of being used for quantitative analysis of those metal ions over selective linear regions. Unfortunately, we do observe a large intercept error for Cu2+.
As shown in fig 3-13, Cu2+ and Co2+ the scatter in the data for the quenching responses also match more closely to a polynomial or exponential equation corresponding to possible upward curvature of the standard Stern-Volmer plot. Such instances of upper curvature has previously been ascribed to distance-based quenching effects.34 Biochemical studies often utilize distance based quenching for positional probing via FRET and such curvatures are a well-known phenomenon of fluorescence quenching.34-42 The specific primary quenching mechanism for Cu2+ or Co2+, whether it be static quenching, sphere of action quenching, or distance dependent quenching, is still an open question since
59 fluorescence lifetimes, time dependent data, and steady state data for this system would be required to fully elucidate the quenching mechanism.
Although some of the metal ions tested have Stern-Volmer equations that are linear with acceptable linear regression values, the quenching mechanism for those ions, Ni2+, Fe3+, and Pb2+, are unclear. Work by Rurack and coworkers suggest that the quenching can occur via an interaction between the metal ion and dye directly1, known as static quenching. While Mancin and coworkers observe static quenching for DDSN,11, 29, 43, 44 when the absorption spectra of the colored metal ion-chelate overlaps with the emission spectra of the dye, energy transfer quenching may occur. However, if the absorption spectra of the complex and emission spectra of the fluorophores do not overlap, then energy release goes through vibrational or lower energy pathways and the static quenching model is favored.
Below we present the Stern-Volmer constants found from the linear equation fits from the graphs above, the higher the value the higher the fluorescence quenching effectiveness of the complex formed on the DDSN:
2 Table 3- 4. Tabulated Stern-Volmer constants, τ0KSV, and regression fits, R for FITC-Terpy-NP1 versus metal nitrate solutions; (20 µg/ml DDSN solution; 1:1 Ethanol:ACN)
Zn2+ Fe3+ Ni2+ Pb2+ Co2+ Cu2+ Fe2+
τ0KSV 0.11 0.35 0.55 0.82 1.48 2.41 0.54 Linear R2 0.93 0.87 0.97 0.98 0.93 0.97 0.97
Looking only at I0/I at 3.0 µM as the criteria for quenching effectiveness, we conclude that the copper-terpyridine complex is the most effective at quenching the fluorescein fluorescent signal with effective quenching order of all tested metal ions in order:
Cu2+ > Co2+ > Pb2+ > Ni2+ > Fe3+ > Zn2+
Furthermore, we observe that Cu2+ and Co2+ are capable of almost completely quenching the fluorescence signal, I less than 90% I0, with a linear range to about 15% of the initial fluorescence. These results demonstrate relative selectivity for this system towards Co2+ and Cu2+ over Fe3+, Ni2+, Pb2+ and especially Zn2+.
60
The quenching mechanism for Cu2+ and Fe3+ is believed to be through energy transfer due to the formation of a colored terpyridine Cu2+/Fe3+ complexes.45 The minimal quenching supports the hypothesis that the. the colorless zinc terpyridine complex does not quench, while the lightly colored complex of terpyridine-Ni2+ exhibits improved quenching. However, the effective fluorescent quenching via the formation of the colorless Pb2+-terpyridine complex suggests a different mechanism for Pb2+. As the Pb2+ has a much larger electron cloud, Pb2+ ions can quench the fluorescence of the fluorophores through electron cloud proximity to the dye without the need for absorption spectra overlap.11, 34 This explanation also reconciles the improved quenching effectiveness observed for the Pb-terpy complex over the similarly colorless zinc-terpyridine complex. Quenching through a non-absorption pathway for Pb2+ has been reported by Mancin and coworkers on thiol-coated DDSNPs with dansyl dye that supports this theory of contact quenching or static quenching.11
61
The metal ion based fluorescent quenching behavior of rhodamine DDSNP surface modified with terpyridine and amino groups was tested in ethanol in an effort to probe different dye interactions and improve solubility in ethanol and water mixtures over FAT-SNP. As a result, the RAT-SNP was synthesized by co-condensing a 1:1:2 ratio of RITC: Terpy-silane: APTES. This ratio, in contrast to the
1:1:1 found in FITC-TerpyNP, improved the dispersability of the nanoparticles into less organic solvents by increasing surface primary amino groups and switching from a fluorescein dye to the more aqueous soluble rhodamine dye. Fig 3-14 and 3-15 shows the fluorescence quenching results for RITC-
TerpyNP(RAT-NP) in ethanol by Ni2+ and Cu2+:
3.5E+06 RITC-TerpyNP 3.0E+06 RITC-TerpyNP + 1.0 2.5E+06 µM Ni2+
2.0E+06
1.5E+06
1.0E+06 Fluorescence Intensity (A.U.) Intensity Fluorescence 5.0E+05
0.0E+00 540 550 560 570 580 590 600 Wavelength (nm)
Figure 3- 14 Fluorescence quenching of RITC-Terpy-NP upon the introduction of Ni2+ up to 3 µM. (20 µg/ml DDSNP; in EtOH)
62
3.5E+06 RITC-TerpyNP RITC-TerpyNP + 1.0 µM Cu2+ 3.0E+06 RITC-TerpyNP + 2.0 µM Cu2+ 2.5E+06 RITC-TerpyNP + 3.0 µM Cu2+ 2.0E+06 1.5E+06
1.0E+06
5.0E+05 Fluorescence Intensity (A.U.) Intensity Fluorescence 0.0E+00 540 550 560 570 580 590 600 Wavelength (nm)
Figure 3- 15 Fluorescence quenching of RITC-Terpy-NP upon the introduction of Cu2+ up to 3 µM. (20 µg/ml DDSNP; in EtOH)
Similar to the fluorescence quenching results observed for FITC-Terpy-NP1, metal ions quenched the
RITC-Terpy-NP emission. Fig 3-14 and 3-15 show fluorescence quenching without significant change in the profile of the fluorescent spectra. Fig x shows the data in Stern-Volmer format:
6 Co2+ 5 Cu2+ Ni2+ 4 Fe3+ Pb2+ /I
0 3
I Zn2+
2
1
0 0 0.5 1 1.5 2 2.5 3 3.5 [Mx+] ; µM
Figure 3- 16 Fluorescence quenching with RITC-Terpy-NP1. Metal ion solutions were made from metal nitrates. (20 µg/ml DDSNP; in EtOH; λex = 525 nm, λem = 575 nm)
The following graphs contain the data from Fig 3-17 split sup so as to offer a clearer representation of the data focusing on key I0/I ranges.
63
y = 0.0988x + 1.0297 1.8 Fe3+ R² = 0.8711 1.7 y = 0.0493x + 1.1587 1.6 Pb2+ R² = 0.2217 y = 0.1201x + 0.953 1.5 Zn2+ R² = 0.9537 /I
0 1.4 I 1.3 1.2 1.1 1 0 0.5 1 1.5 2 2.5 3 3.5 [Mx+] ; µM
Figure 3- 17 Stern-Volmer plot of RITC-Terpy-NP1 versus metal concentration for [Zn2+], [Fe3+], and [Pb2+]; (20 µg/mL DDSNP in Ethanol)
2 1.9 Ni2+ 1.8 y = 0.2923x + 1.0079 R² = 0.9575 1.7 1.6 y = 0.1201x + 0.953 R² = 0.9537 /I Zn2+ 0 1.5 I 1.4 1.3 1.2 1.1 1 0 0.5 1 1.5 2 2.5 3 3.5 [M2+] ; µM
Figure 3- 18 Stern-Volmer plot of RITC-Terpy-NP1 versus metal concentration for [Zn2+], and [Ni2+] (20 µg/mL DDSNP in Ethanol)
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7 Co2+ y = 1.4005x + 1.4435 6 R² = 0.9107 Cu2+ 5 Zn2+ 4 y = 1.2013x + 1.0362 /I 0 I 3 R² = 0.9944 y = 0.1201x + 0.953 2 R² = 0.9537 1 0 0 0.5 1 1.5 2 2.5 3 3.5 [Mx+] ; µM
Figure 3- 19 Stern-Volmer plot of RITC-Terpy-NP1 versus metal concentration for [Zn2+], [Cu2+], and [Co2+]; (20 µg/mL DDSNP in Ethanol)
The Pb2+ data exhibit large scatter (R2 = 0.2217) meaning the RITC-Terpy-NP1 system has a poor linear response to added Pb2+ and obviously cannot be used as a Pb2+ sensor. The linear regression’s for
Zn2+, Ni2+, Cu2+, and Co2+, between 0 to 3.5 µM, yield R2 > 0.9 and the Stern-Volmer equation is a good fit for fluorescence quenching. The good linearity of the curve suggests that the metal ions tested are good static or area of action quenchers and have little distance based quenching effects over the analytical ranges probed
Table 3- below collects the Stern-Volmer constant, Ksv, from the linear fits above.
Table 3- 5.Stern-Volmer constants for RITC-NP1 fluorescence quenching with metal ions Zn2+ Ni2+ Co2+ Cu2+ 0.10 0.30 1.58 1.22
It is clear from the data that Co2+ quenches the nanoparticles most efficiently. In terms of quenching effectiveness, we find:
Co2+ > Cu2+ >> Ni2+ >> Fe3+ > Zn2+
Due to the poor Stern-Volmer fit obtained with Pb2+ that value is not listed. This result is different from FITC-SNP and suggests a higher sensitivity for the system towards Co2+ and Cu2+ as they are the most effective at quenching the fluorescence of the system. Due to the large difference in Stern-Volmer
65 constants between Co2+ and Cu2+ when compared to Zn2+, Fe3+, and Ni2+ the RITC-Terpy-NP1 system is selective towards Cu2+ and Co2+.
When comparing the fluorescence quenching results above for RITC-Terpy-NP1 with FITC-Terpy-
NP, both systems exhibit higher sensitivity towards Co2+ and Cu2+, with the RITC-Terpy-NP system more
2+ effectively quenched with Co , Ksv of 1.58 versus 1.48 while the FITC-Terpy-NP1 system is more
2+ effectively quenched with Cu , Ksv of 2.41 versus 1.21 . The FITC-Terpy-NP system also has a higher
2+ 3+ 2+ sensitivity towards Ni , Ksv of 0.55, and Fe , Ksv of 0.82, compared to the RITC-Terpy-NP, Ni Ksv of
3+ 2+ 0.30 and Fe Ksv of 0.11. The largest difference between the two systems is in their response to Pb
2+ 2+ addition. The FITC-Terpy-NP system shows a linear response to added Pb with a Ksv of 0.82 while Pb is entirely ineffective as a fluorescence quencher in the RITC-Terpy-NP system with poor linear regression and overall response.
In summary, changing from FITC to RITC allowed the DDSNPs to be dispersible in 100% ethanol rather than the more organic ethanol/acetonitrile mixtures. Dispersion in aqueous solvents could be further improved by increasing the amine ratio owing to the amine groups, pKa of ≈ 8, at the surface of the DDSNP, which would be –NH3 at pH 7.
To test this hypothesis, we changed the synthesis of RITC/Terpy/Amino-NP2 to a 1:5 terpyridine- amine ratio versus the 1:2 ratio used in RITC/Terpy/Amino-NP1. The resulting nanoparticles are dispersible in 1:1 ethanol: water solutions and were stable for up to 4 hours as determined by fluorescence stability over the time. The difference in solvent stability between RITC-Terpy-NP1 and RITC-Terpy-
NP2 demonstrate the effectiveness of surface modification on the dispersion of functionalized nanoparticles in solvents.
These results, especially in the cases of Co2+ and Cu2+, highlight the potential for useful levels of selectivity and sensitivity for these systems to be realized since a modest dye change and a chelate:dye ratio change was sufficient to significantly alter both the solubility and the selectivity of these systems.
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3 Future Work
Eventually this type of surface functionalized DDSNP system can be further explored for use with other dyes and surface groups for the selective detection of other targets. By varying the dyes and surface chelate groups, we anticipate being able to further probe the quenching mechanism of DDSNP sensors and gain a better understanding of how nanoparticle sensors compare in both mechanistic details and ranges of analytical utility with other sensors.
Work Cited
1. Calero, P.; Hecht, M.; Martinez-Manez, R.; Sancenon, F.; Soto, J.; Vivancos, J. L.; Rurack, K., Silica nanoparticles functionalised with cation coordination sites and fluorophores for the differential sensing of anions in a quencher displacement assay (QDA). Chem Commun (Camb) 2011, 47, 10599-601. 2. Martinez-Manez, R.; Sancenon, F., New advances in fluorogenic anion chemosensors. J Fluoresc 2005, 15, 267-85. 3. Mahmoudi, M.; Serpooshan, V.; Laurent, S., Engineered nanoparticles for biomolecular imaging. Nanoscale 2011, 3, 3007-26. 4. Coto-Garcia, A. M.; Sotelo-Gonzalez, E.; Fernandez-Arguelles, M. T.; Pereiro, R.; Costa- Fernandez, J. M.; Sanz-Medel, A., Nanoparticles as fluorescent labels for optical imaging and sensing in genomics and proteomics. Anal Bioanal Chem 2011, 399, 29-42. 5. You, L.; Zha, D.; Anslyn, E. V., Recent Advances in Supramolecular Analytical Chemistry Using Optical Sensing. Chem Rev 2015. 6. Chen, L. Y.; Wang, C. W.; Yuan, Z.; Chang, H. T., Fluorescent gold nanoclusters: recent advances in sensing and imaging. Anal Chem 2015, 87, 216-29. 7. Martinez-Manez, R.; Sancenon, F., Fluorogenic and chromogenic chemosensors and reagents for anions. Chem Rev 2003, 103, 4419-76. 8. Moragues, M. E.; Martinez-Manez, R.; Sancenon, F., Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the year 2009. Chem Soc Rev 2011, 40, 2593-643. 9. Yao, J.; Yang, M.; Duan, Y., Chemistry, biology, and medicine of fluorescent nanomaterials and related systems: new insights into biosensing, bioimaging, genomics, diagnostics, and therapy. Chem Rev 2014, 114, 6130-78. 10. Zhou, Y.; Zhang, J. F.; Yoon, J., Fluorescence and colorimetric chemosensors for fluoride-ion detection. Chem Rev 2014, 114, 5511-71. 11. Arduini, M.; Mancin, F.; Tecilla, P.; Tonellato, U., Self-organized fluorescent nanosensors for ratiometric Pb2+ detection. Langmuir 2007, 23, 8632-6.
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12. Zhai, J.; Yong, D.; Li, J.; Dong, S., A novel colorimetric biosensor for monitoring and detecting acute toxicity in water. Analyst 2013, 138, 702-7. 13. Yang, M.; Sun, M.; Zhang, Z.; Wang, S., A novel dansyl-based fluorescent probe for highly selective detection of ferric ions. Talanta 2013, 105, 34-9. 14. Barba-Bon, A.; Costero, A. M.; Gil, S.; Parra, M.; Soto, J.; Martinez-Manez, R.; Sancenon, F., A new selective fluorogenic probe for trivalent cations. Chem Commun (Camb) 2012, 48, 3000-2. 15. Siebert, R.; Tian, Y.; Camacho, R.; Winter, A.; Wild, A.; Krieg, A.; Schubert, U. S.; Popp, J.; Scheblykin, I. G.; Dietzek, B., Fluorescence quenching in Zn2+-bis-terpyridine coordination polymers: a single molecule study. Journal of Materials Chemistry 2012, 22, 16041. 16. Balzani, V.; Ceroni, P.; Maestri, M.; Saudan, C.; Vicinelli, V., Luminescent dendrimers. Recent advances. Top Curr Chem 2003, 228, 159-91. 17. Wang, M.; Abbineni, G.; Clevenger, A.; Mao, C.; Xu, S., Upconversion nanoparticles: synthesis, surface modification and biological applications. Nanomedicine 2011, 7, 710-29. 18. Salinas, Y.; Martinez-Manez, R.; Marcos, M. D.; Sancenon, F.; Costero, A. M.; Parra, M.; Gil, S., Optical chemosensors and reagents to detect explosives. Chem Soc Rev 2012, 41, 1261-96. 19. Arap, W.; Pasqualini, R.; Montalti, M.; Petrizza, L.; Prodi, L.; Rampazzo, E.; Zaccheroni, N.; Marchio, S., Luminescent silica nanoparticles for cancer diagnosis. Curr Med Chem 2013, 20, 2195-211. 20. Atkins, P. W.; Overton, T. L.; Rourke, J. P.; Weller, M. T.; Armstrong, F. A.; Shriver, D., Shriver and Atkins' Inorganic Chemistry. Fifth ed.; Oxford University Press: Great Britain, 2010. 21. Nelson, D. L.; Cox, M. M., Principles of Biochemistry. fifth ed.; W. H. Feeman and Company: 2008. 22. Skoog, D. A.; Holler, F. J.; Crouch, S. R., Principles of Instrumental Analysis. Sixth ed.; David Harris: Belmont, CA, 2007. 23. Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Vyvyan, J. R., Introduction to Spectroscopy. Fourth ed.; Brooks/Cole, Cengage Learning: Belmont, CA, 2009. 24. McNaught, A. D.; Wilkinson, A.; Nic, M.; Jirat, J.; Kosata, B., IUPAC. Compendium of Chemical Terminology, 2nd ed. Blackwell Scientific Publications: Oxford, 2006. 25. Vollrath, A.; Schubert, S.; Schubert, U. S., Fluorescence imaging of cancer tissue based on metal- free polymeric nanoparticles – a review. Journal of Materials Chemistry B 2013, 1, 1994. 26. Sahl, S. J.; Moerner, W. E., Super-resolution fluorescence imaging with single molecules. Curr Opin Struct Biol 2013, 23, 778-87. 27. Freeman, R.; Girsh, J.; Willner, I., Nucleic acid/quantum dots (QDs) hybrid systems for optical and photoelectrochemical sensing. ACS Appl Mater Interfaces 2013, 5, 2815-34. 28. Bau, L.; Bartova, B.; Arduini, M.; Mancin, F., Surfactant-free synthesis of mesoporous and hollow silica nanoparticles with an inorganic template. Chem Commun (Camb) 2009, 7584-6. 29. Bau, L.; Tecilla, P.; Mancin, F., Sensing with fluorescent nanoparticles. Nanoscale 2011, 3, 121- 33. 30. Brasola, E.; Mancin, F.; Rampazzo, E.; Tecilla, P.; Tonellato, U., A fluorescence nanosensor for Cu2+ on silica particlesElectronic supplementary information (ESI) available: experimental procedure; TEM images; NMR, UV-vis and fluorescence spectra; fluoresence titration. See http://www.rsc.org/suppdata/cc/b3/b310582b. Chemical Communications 2003, 3026. 31. Schubert, U. S., Terpyridine-based Materials: For Catalytic, Optoelectronic and Life Science Applications. Wiley-VCH: 2011; Vol. 3, p 52. 32. Hofmeier, H.; El-ghayoury, A.; Schenning, A. P. H. J.; Schubert, U. S., Synthesis and optical properties of (a)chiral terpyridine–ruthenium complexes. Tetrahedron 2004, 60, 6121-6128. 33. Winter, A.; Hager, M. D.; Newkome, G. R.; Schubert, U. S., The marriage of terpyridines and inorganic nanoparticles: synthetic aspects, characterization techniques, and potential applications. Adv Mater 2011, 23, 5728-48. 34. Lakowicz, J. R., Principles of Fluorescence Spectroscopy. Springer US: 2013.
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35. Chen, G.; Song, F.; Xiong, X.; Peng, X., Fluorescent Nanosensors Based on Fluorescence Resonance Energy Transfer (FRET). Industrial & Engineering Chemistry Research 2013, 52, 11228- 11245. 36. Davis, B. W.; Niamnont, N.; Dillon, R.; Bardeen, C. J.; Sukwattanasinitt, M.; Cheng, Q., FRET detection of proteins using fluorescently doped electrospun nanofibers and pattern recognition. Langmuir 2011, 27, 6401-8. 37. Soukka, T.; Rantanen, T.; Kuningas, K., Photon upconversion in homogeneous fluorescence- based bioanalytical assays. Ann N Y Acad Sci 2008, 1130, 188-200. 38. Babu, E.; Mareeswaran, P. M.; Rajagopal, S., Highly sensitive optical biosensor for thrombin based on structure switching aptamer-luminescent silica nanoparticles. J Fluoresc 2013, 23, 137-46. 39. Dorn, I. T.; Neumaier, K. R.; Tampe, R., Molecular Recognition of Histidine-Tagged Molecules by Metal-Chelating Lipids Monitored by Fluorescence Energy Transfer and Correlation Spectroscopy. J. Am. Chem. Soc. 1998, 120, 2753-2763. 40. Kim, S. H.; Jeyakumar, M.; Katzenellenbogen, J. A., Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site- specific fluorophore labeling. J Am Chem Soc 2007, 129, 13254-64. 41. Peng, Z.; Young, B.; Baird, A. E.; Soper, S. A., Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time. Anal Chem 2013, 85, 7851-8. 42. Pihlasalo, S.; Pellonpera, L.; Martikkala, E.; Hanninen, P.; Harma, H., Sensitive fluorometric nanoparticle assays for cell counting and viability. Anal Chem 2010, 82, 9282-8. 43. Rampazzo, E.; Brasola, E.; Marcuz, S.; Mancin, F.; Tecilla, P.; Tonellato, U., Surface modification of silica nanoparticles: a new strategy for the realization of self-organized fluorescence chemosensors. Journal of Materials Chemistry 2005, 15, 2687. 44. Arduini, M.; Marcuz, S.; Montolli, M.; Rampazzo, E.; Mancin, F.; Gross, S.; Armelao, L.; Tecilla, P.; Tonellato, U., Turning fluorescent dyes into Cu(II) nanosensors. Langmuir 2005, 21, 9314-21. 45. Maier, A.; Cheng, K.; Savych, J.; Tieke, B., Double-electrochromic coordination polymer network films. ACS Appl Mater Interfaces 2011, 3, 2710-8.
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4. Quencher Displacement Assays (QDA) and Fluorescence Recovery with Metal Ion Chelated Dye Doped Terpyridine Nanoparticles
4.1 Introduction DDSNPs with surface modification groups have been demonstrated to undergo fluorescence quenching upon chelation of metal ions.1-5 Recent work done by Rurack and coworkers utilizes quenched silican nanoparticles and further introduces freely diffusing ligand group substrates into solution that bind metal ions and compete with nanoparticle surface bound chelate.6 This competition can lead to a recovery of fluorescent signal if the substrate in solution has a higher affinity for the metal ion or a sufficiently large concentration of the substrate is introduced. If the substrate can effectively compete for the metal ion, it will strip the metal ion from the DDSNP surface and the quenched fluorescence will return. The introduction of the competing substrate with resulting fluorescence return upon removal of the metal ion from the NP is known as a quencher displacement assay (QDA).
Figure 4- 1 Diagram for our quencher displacement assay surface
In their work on QDA DDSNPs, Rurack and coworkers probe the mechanism for QDA via fluorescence lifetimes. As the metal is bound to the DDSNP via the surface chelate, the fluorescent lifetime for the NP decreases as distance dependent energy transfer becomes facile. Upon introduction of the competing ligand group substrate, in some cases the analytical substrate of interest, the fluorescence lifetime returns to its native state signaling the removal of the quenching metal ion from the chelating site.
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6 This conclusion is far from conclusive as Cu2+ may not be so easily removed from the tridentate terpyridine by the substrates tested by Rurack and co. due to Cu2+-terpy complexe’s high stability constant.7
Since the introduction of the term QDA in 2011, other systems beyond silica nanoparticles have been adopted as the basis for this scheme for substrate detection. One group in 2014 adopted the QDA scheme for use with quantum dots for the detection of Cd2+ via functionalized CdTe QD which turn off in the presence of phenanthroline.8, 9 However, since our system is silica nanoparticle-based, much like those reported by Rurack and coworkers, we will not further discuss non-silica nanoparticle systems in this thesis.
In chapter 3 we have shown that our synthesized terpyridine functionalized DDSNPs are capable of fluorescence quenching in the presence of metal ions using both fluorescein and rhodamine dyes. Here we demonstrate the FITC-terpy NP system’s potential as QDAs.
4.2 Results and Discussion
For our FITC-terpy-NP system, we chose Cu2+ as the metal ion for quenching due to effectiveness at relatively low concentrations and its linear response in metal ion range to be probed (2.5 uM). The metal ion concentration was set at 2.5 µM to start at an approximately 90% fluorescence quenched DDSNP still in the linear response range to minimize the amount of free Cu2+ present in solution.
The metal bound Cu2+-FITC-terpy-NP1 was crearted by mixing [Cu2+] at 2.5 µM and [DDSN] at 20
µg/ml in 3 ml of 1:1 acetonitrile/ethanol (Figure 4-1). Intensity counts at 525 nm were measured while adding 5 uL aliquots with mixing of five different Cu2+ binding substrates as shown in Fig 4-1.
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12
10
8
0 6 I/I 4
2
0 L-Histidine Epinephrine EDTA Thiocyante L-Lysine
Figure 4- 2. Effect of addition of five equivalents of substrate to a metal ion quenched terpy-DDSN (20 µg/ml). λem = 525nm.
Figure 4- 3 Lewis Structure of the 5 tested substrates. L-Lysine’s binding mechanism is through the carboxylic acid and neighboring amino group with the butyl amino group potentiall wrapping back around onto the metal ion. Epinephrine binds to metal ions through the two adjacent hydroxyl groups. EDTA’s binding mechanism is through rotation across the ethyl moiety between the two tertiary nitrogen atoms to allow the four carboxylic acid groups to bind to the free metal ion. L-Histidine binds to metal ions through the free electrons on the ring moiety and the primary nitrogen. Thiocyanate primarily binds to metal ions through its expanded electron cloud.
Upon addition of any good Lewis base (mono or polydentate) and either remove the Cu2+ from terpy or change the absorbance of bound Cu2+-terpy-DDSNP complex. Either way, we expect disruptions in the quenching mechanism and a recovery of fluorescence signal. Figure 4-2 compiles the data collected on
2+ the Cu -FITC-terpy-NP system where I/I0 represents the signal recovery normalized by the initial
72 fluorescence counts of the Cu2+-FITC-Terpy-NP system. The bar graph shows that L-histidine, thiocyanate, and epinephrine induce a small amount of signal recovery whereas L-lysine barely affects the signal. EDTA is the most effective at returning fluorescence signal, as EDTA is a strong chelator for
18 7 metal ions and is probably the most competitive against terpyridine for the metal ion, Kf CuEDTA = 10 .
However, not even EDTA was able to fully recover the signal, defined as I/I0 = 12.5 or full fluorescence recovery, at 5 equivalents of [EDTA] to [Cu2+]. This phenomenon suggests that the mechanism for signal recovery may not be the complete removal of Cu2+ from the FITC-terpy-NP system.
A closer inspection of the data for L-Histidine and Epinephrine across a variety of concentrations are presented below to further illustrate the quencher displacement behavior.
3 y = 0.0585x + 0.988 Epinephrine R² = 0.881 2.5 L-Histidine y = 0.176x + 0.9767 2 R² = 0.9843 0 I/I 1.5
1
0.5 0 2 4 6 8 10 12 Ratio [Substrate]:[Cu2+]
Figure 4- 4 Effect of substrate on emission recovery for FITC-NP1-Cu2+ (20 µg/ml NP1; 25 µM Cu2+)
As shown in Figure 4-3, both L-histidine and epinephrine restore the fluorescence signal of Cu2+-
FITC-terpy-NP1 as a linear function of concentration. L-Histidine is more effective than epinephrine at returning the fluorescence since it has a larger slope. The exact mechanism for signal recovery is still unknown for these two substrates as it is not clear whether the substrate interacts with the metal by removing it from the terpyridine site or whether it binds to the terpyridine – metal complex changing the
Cu2+-terpyridine complex’s fluorescence quenching effectiveness.
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As even EDTA failed to fully restore the fluorescence at 5 equivalent [Cu2+], it is unlikely that the other substrates are removing the metal ion in our QDA system. We propose here that the substrates L- histidine, epinephrine, thiocyanate, and L-lysine with only a weak affinity compared to EDTA, are unlikely to be able to bind to Cu2+ strongly enough to remove it from the tridentate terpyridine chelate completely and that the substrate is bound to the metal-terpyridine complex changing its fluorescence quenching effectiveness slightly.
In an effort to understand the effect, an excess of L-histidine was titrated into a quenched DDSNP. A continued rise in the signal followed by diminishing returns was observed, shown below:
4.5 4 3.5 3
0 2.5
I/I 2 1.5 1 0.5 0 0 20 40 60 80 100 120 140 160 180 200 Ratio of [L-Histidine]:[Cu2+]
Figure 4- 5 Effect of L-histidine on emission recovery for Cu2+-FITC-terpy-NP1 (20 µg/ml NP1; 2.5 µM Cu2+)
Through Figure 4-4, we demonstrate the diminishing returns associated with continued addition of L- histidine. At around I/I0 > 90, we observe saturation behavior. We were unable to add any more L- histidine to the system without cloudiness appearing past 180 µM [L-histidine]. The maximum I/I0 observed rests around 4.5 meaning a final fluorescence return of 28%, this implies that eventhough high concentrations of L-histidine were observed to slightly alter the quenching mechanism, there is a limit to the amount of L-histidine a single quenching unit can be affected by. This limit supports our theory that
L-histidine is not a strong enough chelator to compete with terpyridine for Cu2+ and thus the fluorescence recovery results from an alternative mechanism from QDA.
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Substrates other than those presented above were tested. Of the substrates tested, phosphates and cyanide ions interacted with the FITC-terpy-NP directly, enhancing the fluorescence signal of the an unquenched, no Cu2+ added, system directly. Cyanide in particular was able to double the fluorescence of fluorescein even at nanomolar concentrations. Azide, citrate, oxalate, and thiocyanate were also tried and did not significantly interact with either FITC-terpy NP or Cu2+-FITC-terpy NP.
4.3 Future Work
Other researchers in the Margerum lab are currently probing the QDA properties of a system with rhodamine B replacing fluorescein as the fluorescent dye due to fluorescein’s observed tendency to interact with various anions. Possible future directions looking into this system involves the testing of the system sensing potential with regards to ADP/ATP, ethylene diamine, catechol and catechol group containing dyes, oligopeptides, DNA bases, and other large molecules that potentially bind to open sites on the terpy-Cu2+ complex in an effort to offer an alternative signal recovery mechanism beyond removal of the metal ion. Work in this area seeks to elucidate the mechanism for signal recovery as more substrate-metal-terpyridine complexes are probed by modifying the strengths of the interactions between the substrate-metal-chelate complexes on the surface of the DDSNP.
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Work Cited
1. Rampazzo, E.; Brasola, E.; Marcuz, S.; Mancin, F.; Tecilla, P.; Tonellato, U., Surface modification of silica nanoparticles: a new strategy for the realization of self-organized fluorescence chemosensors. Journal of Materials Chemistry 2005, 15, 2687. 2. Arduini, M.; Marcuz, S.; Montolli, M.; Rampazzo, E.; Mancin, F.; Gross, S.; Armelao, L.; Tecilla, P.; Tonellato, U., Turning fluorescent dyes into Cu(II) nanosensors. Langmuir 2005, 21, 9314-21. 3. Bau, L.; Tecilla, P.; Mancin, F., Sensing with fluorescent nanoparticles. Nanoscale 2011, 3, 121- 33. 4. Arduini, M.; Mancin, F.; Tecilla, P.; Tonellato, U., Self-organized fluorescent nanosensors for ratiometric Pb2+ detection. Langmuir 2007, 23, 8632-6. 5. Xia, D.; Li, D.; Ku, Z.; Luo, Y.; Brueck, S. R., Top-down approaches to the formation of silica nanoparticle patterns. Langmuir 2007, 23, 5377-85. 6. Calero, P.; Hecht, M.; Martinez-Manez, R.; Sancenon, F.; Soto, J.; Vivancos, J. L.; Rurack, K., Silica nanoparticles functionalised with cation coordination sites and fluorophores for the differential sensing of anions in a quencher displacement assay (QDA). Chem Commun (Camb) 2011, 47, 10599-601. 7. Schubert, U. S., Terpyridine-based Materials: For Catalytic, Optoelectronic and Life Science Applications. Wiley-VCH: 2011; Vol. 3, p 52. 8. Hu, X.; Zhu, K.; Guo, Q.; Liu, Y.; Ye, M.; Sun, Q., Ligand displacement-induced fluorescence switch of quantum dots for ultrasensitive detection of cadmium ions. Anal Chim Acta 2014, 812, 191-8. 9. Wang, K.; Qian, J.; Jiang, D.; Yang, Z.; Du, X.; Wang, K., Onsite naked eye determination of cysteine and homocysteine using quencher displacement-induced fluorescence recovery of the dual- emission hybrid probes with desired intensity ratio. Biosens Bioelectron 2014, 65C, 83-90.
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