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UNIVERSITY OF CALIFORNIA Santa Barbara Characterization of DNA-Stabilized Fluorescent Silver Nanoclusters A Dissertation Submitted in the Partial Satisfaction of the Requirements for Honors in the Degree of Bachelor of Science in Physics By Kira Cocchiara Thesis Advisor: Elisabeth Gwinn Professor of Physics September 2012
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UNIVERSITY OF CALIFORNIA
Santa Barbara
Characterization of DNA-Stabilized Fluorescent Silver Nanoclusters
A Dissertation Submitted in the Partial Satisfaction of the Requirements for Honors in the Degree of Bachelor of Science in Physics
By
Kira Cocchiara
Thesis Advisor:
Elisabeth Gwinn
Professor of Physics
September 2012
2
The Dissertation of Kira Cocchiara is Approved by:
Faculty Mentor Date
Faculty Advisor Date
University of California
Summer 2012
3
Table of Contents
Chapter 1: Introduction………………………………………………………………
I. Abstract……………………………………………………………………
II. Background and Motivation……………………………………………….
Chapter 2: Methods and Materials…………………………………………………..
I. DNA Strands………………………………………………………………
II. Optical Measurements…………………………………………………….
III. Synthesis Procedure……………………………………………………….
IV. Calibrator Dyes for Quantum Yield Measurements……………………….
Chapter 3: Quantum Yield……………………………………………………………
I. Quantum Yield and Responsivity Derivation………………………………
II. Measurement Procedure……………………………………………………
Chapter 4: Results……………………………………………………………………...
I. Salting………………………………………………………………………
II. Quantum Yield Findings……………………………………………………
Chapter5: Conclusion and Outlook…………………………………………………..
4
List of Figures……………………………………………………………………
Appendix: Strand Data…………………………………………………………..
References……………………………………………………………………….
5
Chapter 1: Introduction
I. Abstract
Single stranded DNA oligomers have been shown to stabilize few-atom silver
clusters when in aqueous solution1,2. A number of these ‘AgDNAs’ are fluorescent, with
their emission and excitation wavelengths, quantum yields and chemical lifetimes largely
dependent on the sequence of the stabilizing oligomer3,4. Other important parameters
influencing fluorescence are the ratio of silver atoms to DNA bases, salt content of the
solution, and DNA concentration. Here we report a procedure for fluorescence intensity
optimization and apply this to quantum yield determination of a series of different
AgDNA emitters.
II. Background and Motivation
DNA oligomers have been shown to stabilize inorganic nanoparticles when in an
aqueous solution. Specifically, silver atom clusters less than 30 atoms in size retain
photostable fluorescence in the presence of DNA bases5. The Ag+ ions can bond to either
single or double stranded DNA, binding more preferentially to single unpaired bases, and
do not bind to the phosphate backbone group6. The emissive characteristics of these
fluorescent metal nanoclusters depend on the base sequence of the host DNA strand. The
fundamental characterization of DNA-stabilized fluorescent silver nanoclusters
(Ag:DNAs) can provide insight into rational design of specific fluorophores.
6
Determining the optimal reagent concentrations and emission characteristic discrepancies
in different base sequence provides insight into future designs and uses. Most commonly,
fluorescent Ag:DNA complexes form by binding to cytosine (C) and guanine (G) bases
and not adenine (A) or thymine (T)7. Previous studies have shown that these emitters can
be useful in the fields of bio-labeling8, genetic mutation identification9, or as sensors for
metal ions10. The long-term goal of these optical emitters is to characterize the properties
of AgDNAs and combine them to create a bi-color emitting DNA nanostructure.
Fluorescent silver clusters are bound to DNA using a chemical reduction with
sodium borohydride (NaBH4). Silver nitrate (AgNO3) is typically mixed with a solution
of a relatively short DNA sequence, 6-50 bases long, and then chemically reduced.
Concentrations of the reagents and the DNA sequence involved affect the wavelength of
emitted light from the sample as well as the intensity of that emission. The DNA solution
is typically buffered by ammonium acetate (NH4OAc) to ensure a stable pH of the
solution. The quantum efficiency of these emitters also varies widely from as low as 2%11
to as high as 94%. Adding a salt, such as magnesium acetate (MgOAc), to an AgDNA
solution before chemical reduction has been shown to change the emissive characteristics
of the fluorophore. In solution the 2+ magnesium ion will help stabilize base pairing12.
This is due its screening effect on the negatively charged phosphate backbone of the
DNA strand, which would otherwise stiffen and repel the strands from one another.
Salting DNA solutions is necessary for the assembly DNA nanostructures, such as DNA
origami and DNA nanotubes13. By using these underlying DNA ‘scaffolds’ we can create
interesting dense optical arrays of silver cluster emitters. A high salt concentration during
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the formation of Ag clusters is needed to keep the scaffold intact during this process.
Figure 1.1a shows that over a range of different silver per strand ratios the salted AgDNA
solution is generally the brightest in intensity. Fig. 1.1b demonstrates the usual red shift
in wavelength that is observed from salting AgDNA solutions.
Fluorescence itself is the emission of photons, in the range of ultra-violet (UV) to
infrared (IR) wavelengths. Light, at some starting excitation wavelength, is absorbed by
the electrons in the sample of interest, and then released at a new longer wavelength. The
electrons began in the energetic and vibrational ground state jump to a new state. This
excited state is determined by the overlap of wavefunctions between the original and new
state. Fig. 1.2a demonstrates that an electron beginning in the zero vibrational state will
most probably be found in the second excited vibrational state. The green arrow
a)! b)!
Figure 1.1: a) Plot of Intensity at peak emission wavelength vs. Ag/Strand ratio for both salted and non-salted solutions for AModStrand. b) Fluorescence vs. emission wavelength plot of salted and non-salted solutions of AModStrand at a ratio of 12.5 Ag atoms per DNA strand. The wavelength shift that occurs between the solutions is 76nm.
8
illustrates the loss of energy as light as the electron returns to the energetic ground state
and is referred to as absorbance. When that energy is emitted due to the decay of the
electron back down its ground electronic state in the form of light, it is known at
fluorescence. A shift in the nuclear coordinates occurs when a new vibrational state is
reached in the ground state and results in a wavelength difference between the exciting
light and the emitted light known as the ‘Stokes Shift’.
The wavelength of emitted fluorescence, λem, in pure solutions is independent of
the wavelength of the excitation light, λex, and thus the shape of the emission spectrum is
also independent of the excitation wavelength. The "Kasha-Vavilov" rule states that
radiative transitions are produced from the lowest electron state moving to the
vibrationally excited levels of the ground state. So regardless of how the fluorescent
substance is excited, it always ends up in the same final emissive state since the emission
wavelength is insensitive to the excitation wavelength.
The fluorescence quantum yield gives the probability that the excited state of the
fluorophore will lose its energy by fluorescence emission rather than by any different
method or energy loss. Generally speaking, it is the ratio of photons emitted (through
fluorescence) to photons absorbed. This is an important characteristic for any fluorescent
species and can provide insight into what makes an efficient emitter. The maximum
possible yield of any emitter is 1, or 100%, in which every photon that is absorbed results
in one emitted photon in the form of fluorescent light. Measurements can only be taken
on purified solutions of Ag:DNAs. A solution with more than one species of emitter will
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provide no useful information for the efficiency of that Ag:DNA because photons are
being emitted and absorbed by different clusters. The chemical yield of synthesized
Ag:DNA solutions is not usually 100% and thus needs to have the interesting emitter
separated out from the mixed solution. Our quantum yield measurements are done in a
relative fashion by comparing experimental values to known quantum yield standards,
which will be discussed in further detail in chapter 2.
Figure 1.2: Franck-Condon principle energy diagram and absorption/fluorescence schematic. a) Electronic transitions are most probable between the ground state energy level and the vibrational state in the first excited energy state that gives the best vibrational overlap. This corresponds to minimal change in nuclear coordinates. b) Symmetrical absorption and fluorescence curves, due to potential well shapes, broaden inhomogeneously into two peaks shown by the darker lined curves.
E0:! E1:!
excited state bond length!ground state bond length!
a)! b)!
Figure taken from www.Wikipedia.com
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Chapter 2: Materials and Methods
I. DNA Strands
All oligonucleotide strands were purchased from Integrated DNA Technologies
(IDT) with standard desalting. They were hydrated to the desired concentration with
purified nuclease-free water also purchased from IDT. The strands were stored in the
dark at -20 °C while not in use. All strand sequences are given in Appendix A. Many of
these strands form what are called ‘DNA hairpins’, due to complementary base bonding.
This will form a double-stranded tail and leave a single-stranded silver catching loop at
the head of the nanostructure.
5’ GCT ATG GAC TGC CCA CCC GCA GTC CAT AGC 3’!
G !
C!
C !
G!
C!
|!
A!
C!
C! C!
C!
G !
C!
C !
G!
G !
C!
C !
G!
A !
T!
T !
A!
A !
T!
C !
G!
T !
A!
A !
T!
5’!
3’!
Figure 2.1: Hairpin structure formed by pairing the 5’ and 3’ primes ends of the top sequence. The unpaired loop develops from being the only non-pairing bases present in the sequence. The loop bases: CCA CCC, are bolded in the sequence and the loop.
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II. Optical Measurements
Both fluorescence and absorbance measurements were carried-out with a 100µL,
10mm, sub-micro UV-transparent (Starna) cuvette made from synthetic fused silica
(quartz) specially designed for UV measurements. Spectra were collected using a multi-
purpose spectrometer (Ocean Optics QE 65000). This spectrometer uses a
thermoelectrically cooled charge-coupled device (CCD) array detector. It will accumulate
an electric charge proportional to the intensity of light collected, which is then converted
into a voltage that can be shown as a digital readout on a computer monitor. Fig. 2.2b
indicates the light path taken for excitation of a sample and the data collection performed
by the Ocean Optics system.
Absorbance measurements were made using a Deuterium/Halogen lamp (Ocean
Optics DT-MINI-2-GS) and passed through a quartz neutral density filter (O.D. 1.0) in
order to avoid saturating the array detector with too high of a light intensity. Fluorescence
measurements were performed by exciting the sample with light from a UV lamp for UV
excitation and a high power Xenon arc lamp (Ocean Optics HPX-2000) for visible
excitation. In order to choose a specific wavelength from the broad spectrum of the HPX-
2000, the light was passed through a scanning monochromator (MonoScan 2000) that
was set to the peak excitation wavelength each sample. The sample was removed from
the excitation light path when not taking measurements to prevent bleaching. Fig. 2.2a
shows the absorbance set-up for our measurements. To avoid moving fibers between
measurements for fluorescence and absorbance the DT-Mini was used as the broad band
light source for absorbance measurements instead of the HPX-2000.
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DT-Mini!
1.! 2.! QE65000!
3.!
a)!
2.!
QE65000!
3.!
HPX2000!
Monoscan!
1.!
b)!
Figure 2.2: a) Absorbance measurement set-up. Box 1 contains a broadband light source from which light travels through the red fiber and an f02 optic to reach the sample. Box 2 represents the light-blocking container, which shields the cuvette-containing sample from outside light. Box 3 shows the detector that collects all transmitted light from the sample. The light travels through an optic and the yellow fiber. b) Fluorescence measurement set-up. Box 1 contains a broadband light source. It sends light through the blue fiber to a monochromator, which then travels through the green fiber and an optic to reach the sample. Box 2 holds the sample filled cuvette. Box 3 shows the detector, which collects all emitted light from the sample. This light reaches the detector through another optic and the yellow fiber.
13
For low volume measurements of many samples, the Tecan Infinity 200-Pro
multi-purpose spectrometer was used. Samples were held in a low volume multi-well
plate, 5-50µL per well, with a non-binding surface (Corning 384 well-plate). This multi
sample holding plate allowed for progressive measurements of different silver
concentrations to be made one after another. This system was used for the sole purpose of
taking fluorescence measurements to determine the optimal parameters for forming
specific Ag:DNAs. Variable excitation wavelength from 230-850nm, as well as wide
ranges for light collection make the Tecan the perfect system for discovering the best
parameters for Ag:DNAs.
III. Synthesis Procedure
To prepare a fluorescent solution silver nitrate (AgNO3) is mixed into a solution
of buffered DNA and followed by chemical reduction with NaBH4. The intensity of
fluorescence and other product characteristics depend on the DNA sequence and the
concentrations of all reagents involved. The final mixture will usually contain 10mM
ammonium acetate (NH4OAc), 5µM DNA and a 2:1 ratio of AgNO3 to NaBH4. The
optimal concentrations for the silver and reducing agent are determined by varying the
silver to strand ratio in the final mixture. The Ag/DNA strand ratios of 5, 7.5, 10, 12.5,
15, 20, 25, and 30 correspond to 25, 37.5, 50, 62.5, 75, 100, 125µM final concentrations
of AgNO3 when mixed with a 5µM final concentration of DNA. It is important to wait
approximately 30 minutes after the addition of AgNO3 to reduce the solution in order to
give the silver time to bind to the DNA in solution. Too short a time between additions
will result in a non-fluorescent solution.
14
IV. Calibrator Dyes for Quantum Yield Measurements
In order to determine quantum yield values, and make certain detector
calibrations, a series of organic dyes, purchased from Exciton, were used as comparison
standards with known quantum yields. All dyes were diluted in a solution of 100%
ethanol and had spectrum measurements taken using the OO-Spectrometer. Figure 2.3
shows each of the four calibrator dyes that were used: Rhodamine 101, Cresyl Violet,
Oxazine 1, and HITC1. The useful range of these dyes extends from 500 to 775nm14,
which covers the visible emission area of most of the AgDNAs of interest. The right
curve of each plot in the absorbance spectrum of that dye and the left curve on each plot
in the fluorescence, or peak emission, spectrum. The fluorescence was obtained by
exciting each dye with the peak value of its absorbance spectrum. Table 2.3 shows the
maximum values used for each dye.
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0.12
0.10
0.08
0.06
0.04
0.02
0.00
Ab
so
rba
nce
700650600550500450
Wavelength (nm)
3000
2000
1000
0
Flu
ore
sce
nce
(au
)
Rhodamine 101 AbsorbanceRhodamine 101 Fluorescence
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Ab
so
rba
nce
800750700650600550500450
Wavelength (nm)
1500
1000
500
0
Flu
ore
sce
nce
(au
)
Cresyl Violet Absorbance Cresyl VIolet Fluorescence
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Ab
so
rba
nce
900800700600500
Wavelength (nm)
100
80
60
40
20
0
Flu
ore
sce
nce
(au
)
HITC1 AbsorbanceHITC1 Fluorescence
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Ab
so
rba
nce
800750700650600550500
Wavelength (nm)
200
150
100
50
0
Flu
ore
sce
nce
(au
)
Oxazine 1 Absorbance Oxazine 1 Fluorescence
Figure 2.3: The molecular structure of organic dyes and their absorbance and fluorescence spectrums.
Dye Abs Range (nm) Absorbance Max (nm) Fluorescence Max (nm)
Rh-101 500-565 567.55 589.13
CV 520-610 596.82 620.63
Ox-1 560-650 642.83 659.63
HITC1 650-775 740.02 768.61
Table 2.3: Usable absorbance wavelength ranges along with the peak absorbance and peak fluorescence for each organic dye in nanometers. The solvent for each dye solution was 100% ethanol.
16
Chapter 3: Quantum Yield
I. Quantum Yield and Responsivity Derivation
To measure the quantum efficiency of an unknown Ag:DNA we need an equation
for quantum yield that involves measureable quantities. Our spectrometer can measure
fluorescence and absorbance, which we can related to quantum yield in the following
way. The fluorescence signal reported by the OO-Spectrometer is the converted voltage
measurement from the array’s accumulated electric charge and thus not the actual
fluorescence intensity, If, of the emitting species. The quantum yield, Φ, of a fluorophore
is related to If by the equation:
Eq. 1
The fluorescence intensity is proportional to the measured signal intensity, Sf, by
some spectral response of the detector, which varies by the wavelength detected in the
gratings of the array. We will call this responsivity of the detector r for now, where:
. In accordance with the Beer-Lambert law, absorbance is proportional to
concentration:
Eq. 2
17
Combining these three equations gives a rough relation between quantum yield
and the measureable quantities of the fluorescence signal, the spectral response and the
absorbance:
Eq. 3
Other factors contained in this scaling relation with will be discussed in more detail
below.
To correctly determine the quantum yield of these emitters the responsivity of the
detector must be determined first. The responsivity is dependent upon a single
wavelength determined by the position of the array detector when light is collected. The
light being directed to the detector is coming from the HPX broad-band light source and
output through the monochromator. This means that the responsivity will be related to the
actual intensity of light coming from the monochromator, Imono, and the signal that is
detected by the spectrometer, Smono:
Eq. 4
The signal detected from the monochromator can be found by measuring light
directly output from the monochromator and not through a sample. The output measured
appears roughly as a Gaussian with a spectral width of two nanometers. The wavelength
range of interest for AgDNAs is about 300 to 850nm. By recording the spectrum output
18
from the monochromator every two nanometers in this wavelength range, the wavelength
dependent signal intensity can be determined, as shown Fig. 3.1a.
The actual intensity output from the monochromator can be determined by using
the measured fluorescence excitation signal, Fex, and the measured absorbance spectrum
of different organic dyes. Fex is the fluorescence signal that the detector records, at a fixed
emission wavelength, as a function of the wavelength of the excitation light. This is
illustrated in Fig 3.2 for the dye Cresyl Violet. In Fig. 3.2a, each trace is the full emission
spectrum corresponding to different excitation wavelengths. The peak emission
wavelength is 620.63nm. Taking a ‘slice’ through the emission spectrum at this
wavelength generates the fluorescence excitation signal spectrum, Fex, plotted in purple in
60x103
50
40
30
20
10
0
Monochro
mato
r Sig
nal: S
mono (
au)
800600400200
Wavelength (nm)
Signal From Monochromator
Wavelengths from 300-850nm
Spectral width = 2nm
a)!70x10
3
60
50
40
30
20
10
0M
onochro
mato
r S
ignal: S
mono (
au)
800700600500400300
Wavelength (nm)
Signal from Monochromator Rh-101 Range CV Range Ox-1 Range HITC1 Range
b)!
Figure 3.1: a) Plot of signal detected from output directly from the monochromator every 2nm from 300 to 850nm. The input light source to the monochromator is the HPX-2000 lamp. The output wavelength reported from the monochromator must be corrected for systematic error in the device. b) The peak intensity values from each Gaussian in graph a) fitted together into a continuous signal intensity. This represents Smono. The wavelength range necessary for each dye is overlaid on the plot.
19
Fig. 3.2b. Equations 1 and 2 show that the fluorescence intensity is proportional to the
absorbance, A. So naively one would expect Fex (the detector signal) to have exactly the
same shape as the absorbance spectrum, which is plotted in Fig. 3.2b as a solid orange
line. The reason that the two curves do not have the same shape is that the responsivity of
the detector, r, depends on wavelength. We use this fact to determine the wavelength
dependence of r, by ratioing Fex and A (absorbance). Because each dye has a limited
useful spectral range (see Table 2.3), we had to use 4 different dyes in order to determine
the responsivity, r, over the full range needed in order to measure quantum yields.
In summary, the fluorescence excitation signal can be obtained from taking the
peak intensity value from the fluorescence emission signal only at that specific emission
2000
1500
1000
500
0
Flu
ore
sce
nce
Em
issio
n (
au
)
800750700650600550
Wavelength (nm)
620.63nm
Cresyl VioletFluorescence EmissionExcitation range: 480-680nm
a)! b)!
0.10
0.08
0.06
0.04
0.02
0.00
Ab
so
rba
nce
800700600500400
Wavelength (nm)
1500
1000
500
0
Flu
ore
sce
nce
Excita
tion
(au
)
Cresyl Violet Absorbance Fluorescence
Excitation
Figure3.2: a) The fluorescence emission scan for Cresyl Violet dye for different excitations ranging from 480-680nm. The peak intensity occurs at 620.63nm, with the intensity value for each curve used to create the fluorescence excitation curve. b) This graph shows the absorbance spectrum for Cresyl Violet on the right axis and the fluorescence excitation curve on the left axis. Each point making the fluorescence excitation curve is taken from the peak values of graph a). The absorbance and the fluorescence excitation curves should have close to the same shape.
20
wavelength and plotting it against the excitation wavelength. Setting a fixed emission
wavelength guarantees that the wavelength dependence of the detector is removed; the
responsivity only depends on that emissive output wavelength. The emission wavelength
with the highest intensity is the logical wavelength to choose since it provides the best
spectral signal.
The intensity from the monochromator, Imono, can be found by dividing the
fluorescence excitation signal by the absorbance. Since less light is absorbed the further
the light travels through the sample of interest, to correct for the attenuation of the
exciting light as it passed through the sample we have to divide by a factor of 1-10-A.
Thus the equation becomes:
Eq. 5
An important detail of working with our monochromator is that the wavelength it
actually scans to is different from the wavelength entered into the monoscan program.
The wavelength of the fluorescence excitation must be corrected to account for the
systematic error from the monochromator wavelength selection program. The true
wavelength of exciting light is approximately 2.849nm less than the wavelength
reportedly output by the monochromator. We determined this by comparing the
command wavelength send to the monochromator to the peak wavelength measured by
the QE65000 detector. Since all of these quantities are wavelength dependent it is
extremely important to correctly compare intensity values at the same exact wavelength.
21
The fluorescence excitation spectrum must also be interpolated between the desired
wavelength range in order to divide it by the absorbance spectrum. Because the
absorbance spectrum has 1044 points (the number of pixels in the detector), the
fluorescence excitation must be made to have the same number of data points
corresponding to the same wavelength values to maintain the correct dependency.
Using the equations for Imono and Smono the responsivity can be written out in terms
of measureable quantities:
Eq. 6
Each dye provides a section of the spectral response over a different wavelength range
that was separately
calculated using the ratio of
the signal detected by the
spectrometer to the actual
signal output from the
monochromator. The
proportionality, K, is
different for each dye (and
would also be different for
0.60
0.55
0.50
0.45
0.40
0.35
0.30
Responsiv
ity (
au)
800750700650600550500
Wavelength (nm)
Responsivity in the
range of each dye:
Rhodamine 101
Cresyl Violet
Oxazine 1
HITC1
Figure 3.3: Responsivity of spectrometer over wavelength range of interest for AgDNA measurements. The spectral response was calibrated using each dye separately over its preferred range and then pieced together for a continuous value.
22
the same dye, if we chose to work with a different emission wavelength). For this reason,
we collected data in overlapping spectral intervals and adjusted each dye’s particular K to
‘match’ the curves between different dyes. When pieced together this way, the different
dye data sets provide the wavelength-dependent responsivity that we need for
characterizing Ag:DNAs.
With an expression for the spectral response derived, an equation for the quantum
yield can be more fully expanded upon from that original relation of . There are a
few more parameters to consider that influence the quantum yield. The geometry of the
fibers, the cuvette and the optics involved can be summed up in a constant, G, that will be
the same for every sample as long as the same cuvette is used and the fibers are not
changed or moved. The specific wavelength dependence of the monochromator,
Smono(λex), and the spectral response, r(λex), at the wavelength of excitation for the
sample, λex, are related by the intensity of light output from the monochromator at that
wavelength, Imono(λex):
Eq. 7
Dividing by the light intensity output from the monochromator normalizes out
this intensity factor at that specific wavelength of excitation for a given sample. Since
Imono(λex) cannot be directly measured, the inverse of the previous equation must be used.
The last thing to consider in getting an equation for quantum yield is the fact that photons
23
are collected over a range of wavelengths due to the nature of the array detector gratings
collecting over a range. Quantum yield is defined as the ratio of the total amount of
photons emitted to the total amount of photons absorbed by a sample. Integrating over the
normalized fluorescence signal over the desired wavelength range provides a means of
counting the total number of photons being emitted. Adding all these aspects together
will give an exact equation for finding the quantum yield instead of just a scaling
relation:
Eq. 8
The fluorescence signal must be recorded with a sufficiently low absorbance,
A(λex) ~0.10, to assure that SF,λem(λex) is simply proportional to the absorbance. The
overlay of the fluorescence excitation and the absorbance should be the same shape, after
taking out the responsivity, r.
II. Measurement Procedure
Measuring quantum yield ratios required a series of absorbance measurements to
be recorded at approximately 0, 0.02, 0.04, 0.06, 0.08 and 0.10 along with corresponding
fluorescence data recorded for each successive absorbance measurement. This low
concentration limit was used to assure that all measurements were taken with in a linear
regime. This was necessary for future gradient comparisons between different samples in
order to determine the correct quantum yield. Both fluorescence and absorbance
24
measurements were recorded with five averaged 500 millisecond integrations using the
OO-Spectrometer. Following the equation for Φ, the fluorescence signal was normalized,
by dividing by the spectral response, and the resulting curve was integrated over the full
spectrum of data in order to include all emitted photons. Since the most basic relation of
the quantum yield compares the absorbance spectrum to that integrated and normalized
fluorescence intensity, I plotted the integrated fluorescence versus absorbance and
observed the resulting gradient.
350x103
300
250
200
150
100
50
0
Tota
l N
orm
aliz
ed F
luore
scence (
au)
0.100.080.060.040.020.00
Absorbance
Gradients Cresyl Violet Rhodamine-101 Oxazine-1GreenOrange Red
Figure 3.4: Plot of the total or integrated fluorescence signal, SF, over responsivity vs. the absorbance for three different organic dyes and three different AgDNAs. Each point corresponds to fluorescence and absorbance values taken at different concentrations. Linear fitting was used to produce the slope.
25
Since the slope, m, is the ratio of the integrated fluorescence and absorbance, then
the quantum yield can be written as:
Eq. 9
These are all known value quantities since the gradient can be found by
performing a linear fit on the plot. By comparing the quantum yield of a known standard
emitter, such as an organic dye, the absolute quantum yield of an unknown AgDNA can
be found:
Eq. 10
Here the subscript ‘1’ refers to the Ag:DNA sample and ‘st’ means the particular
dye standard. Since the quantum yield is determined by comparison to a dye, it is very
important to use well-characterized dyes. In the comparison, the geometrical factors
cancel since they are the same value for both the unknown sample and the known
standard. Since both the monochromator signal and the responsivity have intensities that
are dependent on the excitation wavelength, so these values must be considered in the
comparison ratio The responsivity intensities, rAgD and rST, and the monochromator signal
intensities, SmonoAgD and SmonoST, for the AgDNA and the dye standard respectively can be
26
found by taking the y-values corresponding to the specific excitation wavelength used, as
shown in Fig 3.4. Finally, the quantum yield for an unknown AgDNA can be determined
by multiplying the literature quantum yield value of the standard comparison dye by the
ratio value.
(!AgD, SmonoAgD)"
(!ST, SmonoST)"
(!AgD, rAgD)"
(!ST, rST)"
a)" b)"
Figure 3.5: a) The graph shows how values for the exact intensity of Smono at the wavelength of excitation are determined. Each excitation wavelength for the comparison dye, λst, and the AgDNA, ‘λAgD’, corresponds to a y-axis intensity value at that point. b) The responsivity intensity values for the specific excitation wavelengths for the comparison dye and the AgDNA are determined by taking the y-axis value for each different point.
27
Chapter 4: Results
I. Salting
Salting AgDNA solutions with magnesium acetate, MgOAc, before chemical
reduction consistently produces red shifted fluorescence emission. In some cases the
magnesium seems to help stabilize conformations seen in low chemical yield in solutions
without the Mg2+ ions. This can be observed by looking at a particular set of DNA
hairpin sequences called the AModStrands: GCT ATG GAC TGC CCA CCC GCA GTC
CAT AGC. These all have the same loop, which is bolded in the previous sequence, but
different stem lengths: 6, 9, or 12 base pairs.
Figure 4.1 shows silver sweeps for eight different silver concentrations for
solutions of the 12, 9, and 6 base pair AModStrands, both with and without MgOAc. Two
main emission peaks are easily visible for almost all of the stem versions and salt
concentrations. The brightest emission for non-salted solutions occurs at 578nm with a
dimmer emission at 654nm, while the brightest emission for the salted solutions occurs at
654nm with the dimmer emission being near 578nm. At higher concentrations of Ag the
654nm emission can be more clearly seen in the non-salted solutions than at lower
concentrations. The shift between the two peaks is not very large, 76nm, so both
emissions are peaking close enough to each other to shift the maxima in the measured
signal.
Fitting the data to the sum of two Gaussians shows that there are individual peaks
fairly close to 578 and 654nm, consistently. From Fig.4.1 the fitted Gaussian peaks are as
28
follows: b) 561.49nm ± 1.26 and 655.63nm ± 0.234, c) 582.17nm ± 0.692 and 654.99nm
± 1.05, d) 569.28nm ± 0.391 and 640.61nm ± 1.2 and f) 549.35nm ± 1.07 and 653.89nm
± 0.55. The average green peak appears at about 565nm, while the average red peak
appears around 651nm. The magnesium is apparently boosting the chemical yield of the
redder emitter, by promoting the formation of that conformation in solution. The redder
emitter probably requires more stabilization to form in high quantities and thus increases
in brightness in the presence of a high salt concentration.
The fact that the double-stranded stem of an AgDNA can be cut to half the
number of bases and still form the same emitting species is important. It indicates that the
silver cluster is indeed most probably forming on the single stranded loop of the host
DNA strand and not somewhere else on the strand. The dimming of the emission could
be a clue that the stem helps stabilize the cluster formation and thus increases in the
brightness are due to an increased chemical yield in the longer stemmed species.
29
5000
4000
3000
2000
1000
0
Flu
ore
scence (
au)
750700650600550500450
Wavelength (nm)
578nm
AmodStrand_12BaseStemNo Mg2+
654nm
7000
6000
5000
4000
3000
2000
1000
0
Flu
ore
sce
nce
(a
u)
750700650600550500
Wavelength (nm)
654nm
578nm
AmodStrand_12BaseStem2.5mM Mg2+
5000
4000
3000
2000
1000
0
Flu
ore
sce
nce
(a
u)
750700650600550500450
Wavelength (nm)
AModStrand_9BaseStemNo Mg2+
578nm
654nm
4000
3000
2000
1000
0
Flu
ore
scence (
au)
750700650600550500450
Wavelength (nm)
AModStrand_9BaseStem2.5mM Mg2+
578nm 645nm
800
600
400
200
0
Flu
ore
scence (
au)
750700650600550500450
Wavelength (nm)
AModStrand_6BaseStemNo Mg2+
578nm
654nm
1400
1200
1000
800
600
400
200
0
Flu
ore
sce
nce
(a
u)
750700650600550500450
Wavelength (nm)
AModStrand_6BaseStem2.5mM Mg2+
578nm
654nm
a)! b)!
c)! d)!
e)!f)!
Figure 4.1: Emission spectra of AmodStrands, measured with 270nm excitation and all solutions are at 5µM DNA concentration. a) Ag sweep of AmodStrand with a 12 base pair stem and no Mg2+. b) Ag sweep of AmodStrand with a 12 base pair stem and 2.5mM Mg2+. Fitted Gaussian peaks are at 561.49nm±1.26 and 655.63nm±0.234 c) Ag sweep of AmodStrand with a 9 base pair stem and no Mg2+. Fitted Gaussian peaks are at 582.17nm±0.692 and 654.99nm±1.05. d) Ag sweep of AmodStrand with a 9 base pair stem and 2.5mM Mg2+. Fitted Gaussian peaks are at 569.28nm± 0.391 and 640.61nm±1.2. e) Ag sweep of AmodStrand with a 6 base pair stem and no Mg2+. f) Ag sweep of AmodStrand with a 6 base pair stem and 2.5mM Mg2+. Fitted Gaussian peaks are at 549.35nm±1.07 and 653.89nm±0.55.
30
II. Quantum Yield Findings
A total of eight different Ag:DNA emitters have been characterized in terms of
their quantum yield, number of silver atoms, excitation and emission wavelengths and
number of bases in the host DNA strand. Seven of those Ag:DNAs were previously
uncharacterized and had completely unknown properties, the eighth having been
previously studied15 and referred to in this paper as the ‘Teal’ emitter. The number of Ag
atoms in the other DNA-stabilized clusters was recently determined with tandem in-line
high performance liquid chromatography (HPLC) and mass-spectroscopy (MS)16. Not all
Ag:DNAs are easily purifiable due to the fact that based on the length of the DNA
sequence and how it is wrapped around the silver cluster it may be destroyed by the
HPLC column. Quite a few promising strands could not be purified, and therefore no
quantum yield data can be taken, because they could not make through the column whole.
The range of quantum yield values between these emitters is 6-94%, which is an
extremely wide range, indicating many different factors combine to determine the
efficiency of Ag:DNA emitters. Table 4.1 displays the different characteristics for each of
the quantified emitters. ‘Orange2’ is the most efficient Ag:DNA we have seen so far. An
interesting thing to notice is that the other orange emitter, ‘Orange’, has only one more
Cytosine than ‘Orange2’ (shown in red) and only three other bases switched between
Cytosine and Adenine (shown in blue). Orange: TTCCC CACCACCCAGGC CCCGTT
and Orange2: TTCCC ACCCACCCCGG CCCGTT. Both Ag:DNAs emit close to each
other in wavelength, around 630nm, and differ by only two Ag atoms in cluster size, but
have drastically different quantum yields. ‘Orange’ is about 37% efficient, while
31
‘Orange2’ is an amazing 94%. This suggests that base sequence and number of silver
atoms can have a dramatic influence on how well an Ag:DNA fluoresces.
Figure 4.1c seems to show the only thing resembling a trend of any kind in the
different emitter characteristics: the number of silver atoms roughly increases in a
somewhat linear fashion. The redder the emission wavelength of the fluorophore, or
color, the larger the number of atoms in the silver cluster being formed. Filling the gap in
data for species that emit between 590-620nm would be a good starting point for future
research. New and different emitters can only increase our knowledge and thus help fill
in data gaps as well as help show any new trends that have not yet been noticed. Plotting
quantum yield data against other known cluster properties has not revealed a discernible
pattern. Thus, further characterization of these emitters, such as the rigidity of the
complexes, is necessary to gain an understanding of their widely varying fluorescence
efficiencies.
32
a)! b)!
c)!
Figure 4.1: Characterized Ag:DNA data presented in different ways. a) Plot of quantum yield vs. the emission wavelength. b) Plot of quantum yield vs. Ag per number of bases in host DNA strand. c) Plot of the number of silver atoms vs. the emission wavelength of that emitter.
Strand #Bases AbsMax(nm) EmMax(nm) Stokes(nm) Ag# Ag/Base QY Teal 9c 23 403.94 528.87 124.93 11 0.478 0.0657 Greengst 19 486.11 566.67 80.56 10 0.526 0.4351 Green2t5 30 478.32 557.51 79.19 11+10 0.700 0.0960 Orangev3 23 528.87 625.99 97.12 16 0.696 0.3694 Orange2v5 22 570.63 633.65 63.02 14 0.636 0.9391 Redc4gc4 22 562.14 645.88 83.29 15 0.682 0.1259 Red2rock 28 597.59 668.01 70.42 15 0.536 0.7535 Red3ag3 34 589.90 679.43 89.53 15 0.441 0.4061
Table 4.1: Ag:DNA emitters data table. All Ag:DNAs are monomers except Green2, which is a dimer and thus made using two host strands. Teal: TATCCGTCCCCCCCACGGATA, Green: TGCCTTTTGGGGACGGATA, Green2: CGC CCC CTC CGG CGT, Orange: TTCCCCACCACCCAGGCCCCGTT, Orange2: TTCCCACCCACCCCGGCCCGTT, Red: TTCGCCCCCCGCCCCAGGCGTT, Red2: CACCGCTTTTGCCTTTTGGGGACGGATA Red3: GGCAGGTTGGGGTGACTAAAAACCCTTAATCCCC
33
Chapter 5: Conclusion and Outlook
Our results are a promising step in the direction of crafting multi-color optical
arrays and understanding how to better control the formation of useful AgDNA emitters.
We have shown the wide range of efficiency in fluorescent silver clusters stabilized by
DNA oligomers and are closer to understand the parameters that affect emission
characteristics. In the process of finding quantum yields the responsivity of our
spectrometer has been determined and is now useful for any future measurements where
the true fluorescence intensity is needed. More experiments are necessary if we want to
have the ability to predict what properties a particular Ag:DNA will have.
Salting Ag:DNA solutions has led to the production of emitters with new
wavelengths. Understanding how magnesium ions and other salts affect the behavior of
emitters is crucial for realizing optically functional structures. DNA nanostructures, like
DNA origami and DNA nanotubes, require salty conditions for assembly and the
stabilization of the scaffolding of arrays of silver clusters is only possible with the help of
high salt concentrations. Without understanding the results of Mg2+ ions in Ag:DNA
solutions we will never be able to predicts the wavelength of emission and other useful
properties. More strands sequences should be studied with various salt conditions to help
establish a pattern in color behavior. We know there can be an intensity increase as well
as a shift in wavelength, but we need to be able to logically exploit these properties in
order to design more interesting and useful optical arrays.
34
Now that the responsivity of the Ocean Optics Spectrometer has been determined
over the wavelengths we are interested in, we can find the quantum yield for any
purifiable emitter. The real challenge now is to purify more uncharacterized Ag:DNAs to
fill in the gaps in our knowledge. The HPLC column cannot purify some strands as they
get broken up on their way through the column. Purifying more promising strands will
allow us to get a more cohesive set of quantum yield data. The quantum yields that have
been measured so far are across the board in terms of quantum efficiency of Ag:DNA
emitters. We have three different Ag atom clusters all with wildly different quantum
yields: 0.1, 0.4 and 0.75. Resolving this quandary would be a good direction for
continued research. Perhaps overall rigidity is significantly different for each species. A
floppy structure for the low quantum yield emitter, Red, could be due to energy loss
though internal vibrational relaxation instead of photon emission. Modeling structure
possibilities or using cross correlation spectroscopy to measure diffusion coefficients
could be directions for uncovering useful shape information.
Ag:DNAs are an interesting and expanding field with a bright future for new
experiments. Gathering more data on purified solutions, quantum yield values and salt
conditions will really expand our knowledge and hopefully lead to a greater
understanding of how to logically create specific emitters. Creating two color structures
from already characterized species to create new bi-color emitting structures should be
the next step towards more complicated optical arrays.
35
List of Figures
Chapter 1: Introduction
1.1 Salted and Non-salted Emission Plots…………………….
1.2 Franck-Condon Energy Diagram………………………….
Chapter 2: Material and Methods
2.1 Hairpin Structure and Strand Sequence……………………
2.2 Absorbance and Fluorescence Set-up……………………….
2.3 Calibrator Dyes……………………………………………..
Chapter 3: Quantum Yield
3.1 Monochromator Signal Curves………………………………
3.2 Fluorescence Emission and Excitation Curves………………
3.3 Responsivity Curve………………………………………….
3.4 Quantum Yield Gradients for Comparison…………………..
3.5 Coordinates for Responsivity and Monochromator Curves…..
Chapter 4: Results
4.1 Emission Spectra of AmodStrands…………………………...
4.2 Characterized Ag:DNA Data Plots …………………………..
36
Appendix: All Strands Strand Name Base# Sequence Ag/Strand UVEmMax
(nm) Concen.
(µM) QY
3TD 52 GGCGCTTTTGACCTTCTGCTTTATGTCC CCTATTTCTTAATGACTTTTGGCC
15 630 5
AMod18bpstem 42 ATCTTAGCTATGGACTGCCCACCCGC AGTCCATAGCTAAGAT
15 580 5
AMod15bpstem 36 TTTGCTATGGACTGCCCACCCGCAGTCCA TAGCAAA
15 580 5
Astem12bpGCflip 30 GCTATGGACGGCCCACCCGCCGTCCATAGC 20 680 5 AMod12bpstem 30 GCTATGGACTGCCCACCCGCAGTCCATAGC 15 580 5 AMod9bpstem 24 ATGGACTGCCCACCCGCAGTCCAT 12.5 590/650 5 AMod6bpstem 18 GACTGCCCACCCGCAGTC 10 575 5 T5stem12bneck2flip 30 GCTATGGACGGCCCCCTCGCCGTCCATAGC 20 660 5 T5Mod12bpstem 30 GCTATGGACTGCCCCCTCGCAGTCCATAGC 20 670 5 Tloop12bpstem 28 GCTATGGACTGCTTTTGCAGTCCATAGC 7.5 670 5 Tloop12bpstem6C 34 GCTATGGACTGCTTTTGCAGTCCATAG
CCCCCCC 10 660 5
Tloop12bpstemAAbub6C 34 GCAATGGACTGCTTTTGCAGTCCATAG CCCCCCC
10 670 5
8loop6GsMod12bpstem 32 GCTATGGACTGCAGGGAGGGGCAGT CCATAGC
12.5 655 5
CCACCCloop11bpTPettyTail 46 TAGCTGGACTGCCCACCCGCAGTCCAGCTA CCCACCCACCCTCCCA
25 625 5
4Tloop11bpTPettyTail 44 TAGCTGGACTGCTTTTGCAGTCCAGCTACCC ACCCACCCTCCCA
20 625 5
4Tloop12bpCCCACCCl 35 GCTATGGACTGCTTTTGCAGTCCATAG CCCCCACC
12.5 650 5
4Tloop12bp2GAg4t 35 ATGCTGGACTGCTTTTGCAGTCCAGCA TGGAGGGG
15 640 5
GGAGGGloop12bp_2 30 ATGCTGGACTGCGGAGGGGCAGTCCAGCAT 15 585 5 CCACCCloop12bp_2 30 ATGCTGGACTGCCCACCCGCAGTCCAGCAT 15 580 5 0.4351 GS4Tloop (Green) 19 TGCCTTTTGGGGACGGATA 12.5 570 15 GS9Tloop 24 TGCCTTTTTTTTTGGGGACGGATA 7.5 565 5 GS4A 19 TGCCAAAAGGGGACGGATA 12.5 642 5 Patrick9Chp (Teal) 23 TATCCGTCCCCCCCACGGATA 6 530 25 0.0657 Patrick7Chp 21 TATCCGTCCCCCCCACGGATA 7.5 540/670 5 C3GC3AC3GC3A_10bp 36 CATGGACTGCCCCGCCCACCCGCCCAG
CAGTCCATG 25 570 5
C3GC3AC3GC3A_15bp 41 CATGGACTGCCCCGCCCACCCGCCCAG CAGTCCATG
30 570 5
C9T3C9_15bp 51 CCAGCTATGGACTGCCCCCCCCCCTTT CCCCCCCCCGCAGTCCATAGCTGG
25 670 5
[C4T]4_15bp 50 CCAGCTATGGACTGCCCCCTCCCCTCCCC TCCCCTGCAGTCCATAGCTGG
30 530/670 5
[C6TT]3_15bp 52 CCAGCTATGGACTGCCCCCCCTTCCCCCC TTCCCCCCGCAGTCCATAGCTGG
25 660 5
C7TTC7_10bp_CG 36 CATGGACTGCCCCCCCCTTCC CCCCCGCAGTCCATG
15 640 5
C7TTC7_15bp 46 CCAGCTATGGACTGCCCCCCC CTTCCCCCCCGCAGTCCATAGCTGG
15 645 5
C7TTC7_15bp_AAbubble 46 CCAGCTATGGACTGCCCCCCC CTTCCCCCCCGCAGACCATAGCTGG
20 670 5
(C3A)2C3TC3A_15bp 46 CCAGCTATGGACTGCCCCACCCACCCT CCCAGCAGTCCATAGCTGG
25 625 5
(C3A)2C3TC3A_15bpAabub 46 CCAGCTATGGACTGCCCCACCC ACCCTCCCAGCAGACCATAGCTGG
25 630 5
bunny_15bp 59 CCAGCCAGCTATGGGACAGCCCCCAGCCC ACCGCCCCCAGCACACCCATAGCTGGCTGG
25 640 5
10C 24 TATCCGTCCCCCCCCCCACGGATA 10 530/620 5
37
Ag3 (Red3) 34 GGCAGGTTGGGGTGACTAAAAA CCCTTAATCCCC
15 680 5 0.4061
7C 21 TATCCGTCCCCCCCACGGATA 12.5 530/660 5 6CA2 15 CGCCCACCCCGGCGT 10 605 5 6CA6 15 CGCCCCCCCAGGCGT 10 565/690 25 6CT6 15 CGCCCCCCCTGGCGT 10 570/692 25 C6 6 CCCCCC 12.5 730 80 g6 8 TGGGGGGT 7.5 705 100 6CT5 (Green2) 15 CGCCCCCTCCGGCGT 10 600 25 0.0960 CCCCTT 15 CGCCCCCCTTGGCGT 10 560/710 15 C9loop18bpRight 45 AAAGCCCCATTCATGAGACCCCC
CCCCTCTCATGAATGGGGCTTT 15 618 5
C9loop18bpLeft 45 TCTCATGAATGGGGCTTTCCCCC CCCCAAAGCCCCATTCATGAGA
15 630 5
Ritchie475 12 TTTTCCCCTTTT n/a n/a 5 T4C3T4 11 TTTTCCCTTTT n/a n/a 5 Dickson660 12 CCCATATTCCCC 5 668 15 Dickson680 12 CCCTATAACCCC 5 690 25 Dickson710 12 CCCTAACTCCCC 10 712 15 Petty770 16 CCCACCCACCCGCCCA 12.5 770 20 Petty900 16 CCCACCCACCCACCCG 15 700/900 25 C4GC3A 21 TTCGCCCCCCGCCCAGGCGTT 12.5 600 15 C4GC4A (Red) 22 TTCGCCCCCCGCCCCAGGCGTT 12.5 655 25 0.1259 C5GC5A 24 TTCGCCCCCCCGCCCCCAGGCGTT 10 600 15 C4GC5A 23 TTCGCCCCCCGCCCCCAGGCGTT 10 630 5 C4GC4GC4A 27 TTCGCCCCCCGCCCCGCCCCAGGCGTT 10 665 5 C3GCGCG4A 23 TTCGCCCCGCGCCCCCAGGCGTT 10 640 5 C4AC3A 21 TTCGCCCCCCACCCAGGCGTT 12.5 655 5 C4AC4A 22 TTCGCCCCCCACCCCAGGCGTT 12.5 600 5 AC4AC4AC4A 28 TTCGCCACCCCACCCCACCCCAGGCGTT 12.5 635 5 MostlyC's 21 TTCCCACCCACCCGGCCCGTT 12.5 645 25 MostlyC's_V2 23 TTCCCCACCACCCAGGCCCCATT 12.5 660 5 MostlyC's_V3 (Orange) 23 TTCCCCACCACCCAGGCCCCGTT 12.5 630 15 0.3694 MostlyC's_V4 23 TTCCCCGCCACCCAGGCCCCGTT 10 630 5 MostlyC's_V5 (Orange2) 22 TTCCCACCCACCCCGGCCCGTT 10 630 5 0.9391 MostlyG's 25 TTACGGTGGGTATTGGGGAGGGATT 12.5 640 5 Rockstar (Red2) 28 CACCGCTTTTGCCTTTTGGGGACGGATA 12.5 670 15 0.7535
All solutions prepared with 10mM NH4OAc (Patrick9Chp with 40mM) and 2:1 Ag to NaBH4 ratio
38
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