OPTIMIZATION OF LIPOSOME-ENHANCED HIGH
THROUGHPUT BIOASSAYS
A thesis
presented to the faculty of the Graduate School
of Cornell University
in partial fulfillment of the requirements of the degree of
Master of Engineering
by
Jessica Sailor
January 2007
© Jessica Sailor, 2006
ii
Abstract
High throughput analysis is important for the rapid screening or quantitative analysis of
large number of samples Microtiter plates provide an excellent platform for high
throughput assays and have been used for decades in immunological and nucleic acid
assays. Work focused here on the adaptation of nucleic acid bioassays with liposome
amplification in a microtiter plate format with two main objectives: (1) optimization of
protein-liposome coupling chemistry and (2) optimization of DNA immobilization in
microtiter plates. The protein streptavidin was coupled to COOH-labeled liposomes
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide*HCl (EDC) chemistry. This
procedure was optimized with respect to liposome encapsulation efficiency (total lipid
concentration over encapsulated dye concentration) and binding efficiency. For the
optimization of DNA probe immobilization in microtiter plates, five different plate types
were investigated; medium binding, high binding, NeutrAvidin coated, and two different
types of amine binding. Assay protocols for each plate were developed in addition to the
ability to immobilize DNA probes and the effect on both the target sequence
hybridization and liposome binding. In all cases, data was recorded using a fluorescence
plate reader or dynamic light scattering.
Optimal conditions for the coupling of streptavidin to COOH-labeled liposomes
were determined as 66% liposomes in MES buffer at pH 7 and a streptavidin
concentration of 0.05 mol% of the liposome phospholipid concentration. A coupling time
of 15 minutes was sufficient. In the case of the DNA probe immobilization in microtiter
plates, a probe concentration of 1 to 50 nM enabled target sequence hybridization in all
plates, with a maximum signal to noise ratio (S:N) at about 50 nM. In the case of DNA-
iii
labeled liposomes only the NeutrAvidin plates generated a signal, with a maximum S:N
of about 400:1. In contrast, for streptavidin-labeled liposomes, the NeutrAvidin plates
generated no signal, but the high-binding adsorption plates generated a maximum S:N of
about 80:1 (in all cases at 50 nM of target sequence). The covalent binding plates
generated a S:N of only 5:1 to 10:1. Following from these results, it is recommended that
the NeutrAvidin plate be used with DNA-labeled liposomes to perform a specific test and
that the high binding adsorption plate be used with streptavidin-labeled liposomes to
achieve a universal assay format.
iv
Acknowledgements
I would like to thank my advisor, Professor Antje Baeumner, for all of the support and
help she has given me in completing this project, as well as being a wonderful person to
work with, as both a teacher and an advisor.
Also, Dr. Katie Edwards, who taught me everything I know about laboratory equipment
and procedures, and without whom I would still be standing in the lab, looking like a deer
caught in the headlights of an oncoming truck.
My thanks to the Department of Biological and Environmental Engineering at Cornell
University, as well as the Office of Student Support, for making my experience at Cornell,
both as an undergraduate and graduate student, memorable and fulfilling. Without the
faculty and staff of these departments, my time at Cornell would have been much less
rich, and this project more difficult for a lack of people to either talk to about my work, or
distract myself from it.
Lastly, my parents and friends, who encouraged me to pursue this degree, and then forced
me to do my work when I didn’t want to.
v
Biography
Jessica Sailor is from the suburbs of Philadelphia, Pennsylvania, where she has always
lived. She has pursued both a Bachelor of Science and Master of Engineering degree in
Biological Engineering at Cornell University, Ithaca, NY, where her main interests were
in biomedical applications of engineering, especially as relates to the pharmaceutical
industry. Outside of academics, she enjoys reading, cooking, and spending time with
friends and family.
vi
List of Figures
Figure 1. Generalized DNA-sandwich assay……………………………………………..3
Figure 2. Cutaway cartoon of a generic liposome………………………………………..7
Figure 3. Representation of strip assay format…………………………………………...8
Figure 4. Cartoon of universal liposome assay……………………….………………..…9
Figure 5. Schematic of EDC coupling chemistry…….………………………………....14
Figure 6. Streptavidin liposome binding to a biotinylated plates…………………….…23
Figure 7. Cartoon depicting hybridization complexes for different microtiter plates…..26
Figure 8. Cartoon depicting hybridization complexes for different microtiter plates and
streptavidin binding for biotinylated reporter probes………………………..…..26
Figure 9. Comparison of different microtiter plates in a liposome-enhanced DNA
sandwich assay………………………………………………………………..….28
Figure 10. Comparison of different microtiter plates at the target hybridization step in a
liposome-enhanced DNA sandwich assay…………….……………………..…..29
Figure 11. Cartoon depicting pre-hybridization assay format using adsorption capture
probe immobilization…………………………………………………………….30
Figure 12. Comparison of assay formats for liposome-enhanced sandwich assay to
determine viable procedures………………………………………………..……31
Figure 13. Comparison of different microtiter plates in a universal liposome-enhanced
DNA sandwich assay ……………………………………………………………33
vii
List of Tables
Table 1. DNA probes used………………………………………….…………..……….13
Table 2. Blocking solutions and outline of investigation strategy....................................19
viii
List of Abbreviations
BSA
Bovine Serum Albumin
COOH
Carboxyl
DLS
Dynamic Light Scattering
DNA
Deoxyribonucleicacid
DPPC
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DPPE
N-glutaryl 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine
DPPG
1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt
EDC
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl
HSS
HEPES Sucrose Saline
MES
2-(4-Morpholino)-Ethane Sulfonic Acid
OG
n-octyl-β-D-glucopyranoside
PBS
Phosphate Buffered Saline
PEG
Poly-ethelyene-glycol
PVP
Polyvinyl Pyrrolidone
RNA
Ribonucleicacid
SAM
Self-Assembled Monolayer
S:N
Signal to Noise Ratio
SRB
Sulforhodamine B
TAMRA
Tetramethylrhodamine
TBS
Tris Buffered Saline
TE
10 mM Tris-Cl, 1 mM EDTA
TEG
Triethylene Glycol
ix
Table of Contents
Abstract……………………………………………………………………………………ii
Acknowledgements……………………………………………………………………….iv
Biography……………………….………………………………………………..………..v
List of Figures…………………………………………………………………………….vi
List of Tables………………………………………………………………...…………..vii
List of Abbreviations…………………………………………………………..………..viii
Table of Contents………………………………………………………….…………..….ix
Chapter 1 – Introduction……………………………………………………….………….1
1.1 High Throughput..……………………………………………………….…….1
1.2 DNA Assays……………………………….…………..………………..……..2
1.3 DNA Immobilization………………………………………………….…..…..3
1.4 Streptavidin/Avidin…………………….………………..…….………………5
1.5 Liposome-DNA(RNA) Analysis……….……………………………..……....5
1.6 Universal Assay…………………………………………………..……..…….7
Chapter 2 – Design…………………………………………………………………….…..8
Chapter 3 – Materials and Methods…………………………………………………..….10
3.1 Materials…….………….………………………………..…………….…….10
3.2 Methods………………………………………………………..……………..10
3.3 Liposomes………………………………………..…………………………..10
3.4 Labels……………………………………………………………………..….11
3.5 Optimization of Streptavidin Liposomes…….…………………………....…12
x
3.6 Evaluation and Comparison of Microtiter Plates…………………..….…..…14
3.7 NeutrAvidin plates…………………………………..……………………….14
3.8 Unlabeled plates……………………………………..……………………….15
3.9 DNA-BIND plates……………………………………………………..…….15
3.10 Reacti-Bind Maleic-Anhydride plates……………………..….……..….….16
3.11 Blocking Solutions………………………………………...………………..16
3.12 Mixed Assay Format………………………………………………..………17
3.13 Universal Assay…………………………..……………………….………..18
Chapter 4 – Results and Discussion ……………………………………………………..19
4.1 Optimization of Streptavidin Liposomes………………………………...…..19
Chapter 5 – Results and Discussion ……………………………………………………..23
5.1 Evaluation and Comparison of Microtiter Plates…………………………….23
Chapter 6 – Conclusions and Future Works……………………………………………..34
6.1 Optimization of Streptavidin Liposomes…………………………...………..34
6.2 Evaluation and Comparison of Microtiter Plates………………….……..…..35
References……………………………………………………….……………………….37
xi
Chapter 1– Introduction
1.1 High Throughput
The need to analyze hundreds to thousands of samples on a routine basis has led
to the development of many high throughput methods in pharmaceutical, analytical, and
basic research. One of the most often used platforms has been the 96-well microtiter
plate, for which robots, pipets, and other equipment have been designed to further
increase the efficiency of these plates. In fact, the need for even higher throughput is
often based on multiples of the 96-well plate, such as 384-well, 768-well, etc. formats1.
While there are many effective detection methods available, such as SDS-PAGE,
Western-Blot, and gravitational columns, most of these methods are both time consuming
and labor intensive2,3. While microtiter plates cannot replace these technologies in all
circumstances, they are often simpler to use, needing only common laboratory equipment
in most instances, and taking less time than larger-scale assays. Part of the convenience
of 96-well microtiter plates is that they are available modified for a wide variety of
functions. Some of these functionalizations are chemical4, such as the immobilization of
a DNA probe, as discussed below, and some of the functions are physical, such as
adaptation to run vacuum-driven columns through each well2. Because of these
functionalizations, as well as the speed and convenience provided by robots and pipets,
an assay time can be reduced from twelve or more hours to five or six. Also, 96
experiments can be run in parallel on one of these plates, and with robots and multichannel pipets, physical variations between the experimental procedure between each
well can be reduced, decreasing the variation between the results of each experiment2.
1
Another benefit of microtiter plates is that they use small volumes of reagents,
generally between 100 and 300 µL. This is a vast improvement over many conventional
assays, which often use 2 – 3 mL of a reagent5 or more in the case of most
chromatography procedures. The reduction in volume reduced the cost of the assay,
especially those that use expensive reagents. Also, because the sample volume can be
very small, e.g. needle biopsies, microdissected tissue slices, cytological samples, etc.,
methods that only require small volumes of sample can help to conserve the sample as
well as provide more accurate results6.
1.2 DNA Assays
DNA has become an important part of various analysis and delivery systems in
recent years. In analysis, many of the applications are for detection, both of pathogens
and contamination in food and the environment7. Nucleic acids are perfect for detection
assays because they are highly specific, being able to determine which strain of a species
a bacterium belongs to. DNA is a more robust nucleic acid than RNA, and is therefore
more suitable to an assay8. In many of these assays, a short sequence of DNA, called a
probe, is used to detect a longer sequence of DNA or RNA, called the target, which is
generated by the organism in question. The probe is generally immobilized on some
surface and then mixed with the target to allow them to hybridize. At this point, a signal
needs to be generated and measured. This signal is usually generated by adding a second
DNA probe with a modification, such as the attachment of a fluorescent molecule that
allows the detection of the probe that is bound to the target once the excess has been
washed out4. This combination of probe-target-probe is often called a DNA sandwich
assay. A general diagram of this assay is shown in Figure 1.
2
Signal
Reporter
Probe
Target
Capture
Probe
Immobilization Surface
Figure 1. Generalized DNA-sandwich assay. In this assay, a DNA-capture probe is
immobilized on an appropriate surface, the target sequence of DNA or RNA is hybridized
to the capture probe, and then a reporter probe is hybridized to a different location on the
target. The reporter probe is labeled with a signal – typically a dye molecule, an enzyme
with a product that can produce an electrochemical signal, or a liposome. Drawing not to
scale.
Sandwich assays are not the only method of nucleic acid target detection using a DNA
probe8, but these other methods are outside the scope of this thesis.
1.3 DNA Immobilization
For a DNA sandwich assay, several methods for immobilizing the capture probe,
the probe which detects the target sequence, can be used. The most commonly used
methods are adsorption, covalent bonding, biotin-streptavidin interactions, and self-
3
assembled monolayers4,9. Self-assembled monolayers (SAM) frequently use a gold
surface and allow specific chemicals, such as thioglycolic acid or alkane thiols, to bind to
the gold in a monolayer, leaving an array of functional groups, typically thiol, amine or
carboxyl groups, facing away from the gold. These functional groups then bind to
modified DNA to make a covalent bond9,10. In essence, a SAMs typically bonded to the
surface via adsorption or physic/chemisorption represents a specific variation of
adsorption methods to functionalize a surface prior to covalent bonding of the DNA
probe to the surface.
In adsorption, DNA adsorbs to the surface of the plate, aided by the chemical
make up of the immobilization solution. The solution typically contains chaotropic salts
and one or more polymers such as poly-ethelene-glycol (PEG), which help the DNA to
come out of solution, adsorbing to the styrene surface in an orientation that allows it to
associate with the complementary DNA or RNA strand. Křížová and colleagues
determined that carboxyl groups on the plate are also essential, and adsorption correlates
directly with the number of carboxyl groups available, peaking at roughly 2.6% carboxyl
groups. The salt is present in a low concentration, as this dehydrates the DNA, forcing it
into a more favorable conformation, but high concentrations of salt, the salt competes
with the DNA for the surface charges, compromising adsorption11. In adsorption, the
DNA binds to the surface along its backbone, putting it into a horizontal orientation with
the bases exposed. Because of this rigid immobilization, the complementary DNA does
not actually hybridize to the immobilized DNA, but still associates with it. Berney and
Oliver found that the thickness of the immobilized layer is about 0.7 nm, and some of the
4
DNA does wash away with repeated washings, resulting in approximately a 35% final
coverage of the surface4.
Covalently attached DNA is usually attached by an amine-linkage, which is done
by functionalizing either the DNA or the plate with an amine-group and then the other
member with a chemical such as N-hydroxysuccinimide ester, which interacts with the
amine to form a covalent bond. Because the DNA is modified on either the 5’ or 3’ end,
the DNA can orient vertically when it is immobilized. Because this immobilization is by
a covalent bond, the immobilized DNA does not wash away with repeated washings. The
spacer between the plate and the immobilized DNA provided by the linkage varies in
thickness depending on the exact chemicals used and any folding that may occur, was
estimated to be as much as 1.14 nm by Berney and Oliver. They also determined the
coverage on the plate to be approximately 45% of the functional groups, with a minimum
spacing of 2 nm between DNA strands4.
Streptavidin or avidin can also be used in immobilization. These proteins are
explored in greater depth below. Generally, avidins are tetrameric proteins that bind up
to four biotin molecules so tightly as to be essentially an irreversible reaction12. For
DNA immobilization, avidin is bound to the plate, generally covalently, and biotinylated
DNA is allowed to bind to the avidin. Because avidin is a protein, it is spacially more
constrained than a covalent bond, and so the density of DNA after immobilization is
about four times less than that with covalent bonding. However, avidin also provides a
larger spacer, being approximately 6.5 nm thick on the plate. This extra distance from
the plate can help promote hybridization of the probe to the target. If avidin is bound
5
irreversibly to the surface, than the concentration of DNA immobilizied will not decrease
with washing4.
1.4 Streptavidin/Avidin
Streptavidin and avidin are similar proteins that are obtained from Streptomyces
avidinii and egg white, respectively. The molecular weights is about 58 kDa and 67 kDa,
respectively. Both are tetrameric proteins with one biotin binding site per monomer. The
binding affinity is extremely high for a protein interaction (1015 M-1) and is thus
considered irreversible. Avidin and streptavidin are highly stable proteins, and have
several sites to which modifications can be attached without affecting their ability to bind
biotin13. Pierce manufacturing promotes a product called NeutrAvidin, which is avidin
that has been deglycosylated to reduce non-specific binding of lectin14.
1.5 Liposome-DNA(RNA) Analysis
Liposomes are vesicles consisting of a phospholipid bilayer with the hydrophobic
chains creating a hydrophobic layer and the hydrophilic head groups oriented toward the
extravesicular solution and toward the inner cavity. See Figure 2 for a generalized
diagram of a liposome. The external surface of the liposomes can be modified in several
ways, either by modifying the head groups directly or by adding other molecules to the
membrane composition itself. For example, cholesterol is often added to the membrane
composition to promote stability, and different types of phospholipids may be used for
the characteristics intrinsic in their headgroups. The head groups are modified by the
attachment of functional groups, such as carboxyl or amine groups, which can later be
used to attach other molecules, such as DNA, antibodies, or other proteins. The content
of the inner cavity can also be modified during the formation of the liposomes. Many
6
different types of hydrophilic molecules can be encapsulated, including enzymes, DNA,
signal molecules such as dyes and electrochemical markers, and some pharmaceutical
agents15.
Inner Cavity
Figure 2. Cutaway cartoon of a generic liposome with the lipids represented in blue and
the hydrophilic head groups of the phospholipids represented in yellow. The inner cavity
can be filled with substances such as dyes, proteins, and nucleic acids, and the outer head
groups can be labeled with functional groups, proteins, nucleic acids, etc. Drawing is not
to scale.
Liposomes with DNA attached to the surface can be used to generate the signal in
a DNA assay, and yield accurate results faster than conventional methods such as
Northern Blots. The original format of these assays involved allowing DNA-tagged
liposomes containing the purple-pink dye sulforhodamine B to hybridize with target
DNA in solution, and then 10 µL of this solution was applied to the bottom of a thin
nitrocellulose strip and allowed to migrate up the strip. On the strip, a capture probe is
immobilized in a thin band, and as the liposome-target complex migrates up the strip, it
will bind to this zone. The strip is flushed with buffer, and the test is positive if a pink
7
band remains on the strip16 (See Figure 3). This assay can be modified to work on a 96well microtiter plate as well as in microfluidics devices. The signal generated can be
further amplified by lysing the liposomes using a surfactant, which releases the dye inside
in the case of fluorescent dyes such as SRB, the dye will not be self-quenched anymore
resulting in a significantly enhanced fluorescence signal15.
Figure 3. Representation of strip assay format. The strip before use is show on the far
left with the capture zone represented in faint blue. A drop of DNA-labeled liposomes
that have already been hybridized with the target is placed on the bottom of the strip.
Buffer is then added to the bottom of the strip, washing the liposomes toward the top. If
the test is positive, the target binds to the probe in the capture zone, leaving a bright pink
line.
1.6 Universal Assay
In addition to the liposome-DNA assay described above, in which the liposome is
tagged with a specific DNA probe to detect a specific target, a more universal liposome
can be generated. In this assay, the liposome is tagged with a protein, such as
8
streptavidin or protein G, which then either detects a probe labeled with a complementary
molecule, such as biotin, that is hybridized with the target, or can detect the target
directly. A diagram of this assay is shown below in Figure 4. These assays are called
universal because only one type of liposome is needed to detect a variety of different
targets, which can save time and cost on the production of the liposomes15,17.
Liposome
Streptavidin
Reporter Probe
Biotin
Target
Capture Probe
Immobilization Surface
Figure 4. Cartoon of universal liposome assay using Streptavidin-tagged liposomes. A
capture probe is immobilized to a surface, the target is allowed to hybridize followed by a
biotinylated reporter probe. Then, streptavidinylated liposomes are added and bind to the
biotin. The liposomes can be lysed to release any dye they contain, amplifying the signal.
Drawing is not to scale.
9
Chapter 2: Design
The currently performed liposome-DNA microtiter plate assay utilized
NeutrAvidin coated plates (see Figure 1a in Chapter 3). It provides very reliable data and
low limits of detection. However, it has two drawbacks: (1) each plate is expensive
(about $32), and (2) liposomes tagged with streptavidin cannot be used in the plates (data
are shown in chapter 5) due possibly to interactions between biotinylated probes,
NeutrAvidin and streptavidin on the surface and the liposome, respectively. Thus, the two
main objectives of this thesis are (1) to determine a less expensive microtiter plate and
immobilization protocol for DNA oligonucleotides and (2) to prove its use with
streptavidinylated liposomes. The latter point is of importance so that universal liposomes
can be used in an assay, i.e. one type of liposome can be used for a variety of different
analytes, as described in more detail above.
Experiments were thus performed on a variety of microtiter plates to determine
the dynamic range of detection and the maximal signal to noise ratio (S:N) generated by
each plate. These two parameters were considered the most important characteristics of
the plate in determining both how effective they were and whether they would be a viable
replacement for the currently used NeutrAvidin plates. The NeutrAvidin coated plates
were used as a reference and all other plates were compared to it. In the event that a plate
was found which had comparable or better performance, a more in-depth review of the
cost of running the assay on that plate would be done to determine if it would cost less
than the current assay does. It was theorized that by using plates with different surface
modifications, costs might be reduced and streptavidin-tagged liposomes might be a more
feasible option. In order to test these plates, the currently performed liposome-DNA
10
assay was run on each plate selected, modifying the procedure only to account for the
different immobilization strategies required for the different plate surface modifications.
To determine how each plate, including the NeutrAvidin coated plates, would perform
using streptavidin-tagged liposomes, the same procedures were used, substituting a
biotinylated probe and streptavidin-tagged liposomes for the DNA-tagged liposomes used
in the original assay.
Two different adsorption plates and two different covalent bonding plates were
tested. One adsorption plate was an unmodified 96-well polystyrene plate, costing
approximately $0.50 each. The other plate was irradiated to functionalize the surface
with carboxyl groups. These plates cost about $3 each. Both of the covalent binding
plates bind amine-labeled DNA, but are supplied by different companies and thus have
different surface chemistry. These plates cost $8 and $15 each. These plates were used
because they were readily available, and used common surface modifications.
Additionally, a pre-made immobilization solution was purchased to aid in the adsorption
of the DNA to the adsorption plates.
11
Chapter 3 – Materials and Methods
3.1 Materials
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)], sodium salt (DPPG), N-glutaryl 1,2-dipalmitoyl-sn-glycero3-phosphatidylethanolamine (DPPE), and the extrusion membranes were purchased from
Avanti Polar Lipids (Alabaster, AL). Sulforhodamine B (SRB) was purchased from
Molecular Probes, Inc. (Eugene, OR) DNA Reacti-BIND immobilization solution was
purchased from Pierce (Rockford, IL) All other reagents used in these experiments were
purchased from VWR (Bridgeport, NJ) The DNA sequences listed in Table 1 were
purchased from Operon Biotechnologies, Inc. (Alameda, CA). NeutrAvidin Reacti-Bind
96-well microtiter plates and Maleic Acid Reacti-Bind (amine binding) 96-well microtiter
plates were purchased from Pierce (Rockford, IL), medium binding plates from Thermo
(Milford, MA), high binding plates from Greiner Bio-One (Monroe, NC), and DNABIND (amine binding) plates from Corning (Acton, MA).
3.2 Methods
3.3 Liposomes
Liposomes were provided by Dr. Katie A. Edwards. They were synthesized using
a standard protocol18 using the reverse phase evaporation method. They were
characterized with respect to phospholipid content using a Bartlett Assay19 and size using
dynamic light scattering (DLS). Liposomes contained 150 mM sulforhodamine B (SRB).
SRB can be detected using 540 nm and 590 nm as excitation and emission wavelengths,
respectively. Liposomes were either tagged with a reporter probe binding directly to the
12
DNA target sequence, or were labeled with streptavidin capable of binding to
biotinylated reporter probes. All sequences used in this assay are given in Table 1.
Table 1. DNA probes used. Capture probes were immobilized onto the plates, The
biotinylated reporter probes were used in the universal assay and the TEG labeled
reporter probes were imbedded into liposomes for use in the specific assay. TEG is a
hydrophilic triethylene glycol spacer between the liposome and the probe.
Name
Sequence (5’ – 3’)
Label
Target
ATAAATACGCGGACATCTTGTCTTCTCTTCCCGATATTTCTAG TAMRA (5’)
(540/590 nm
excitation/emission)
Capture
CTAGAAATAACGGGAAGAGAA
Probe
Fluorescein- CAAGATGTCCGCGTATTTAT
6-Fluorescein~Q
labeled
(3’)
Capture
(485/528 nm
Probe
excitation/emission)
Biotinylated CTAGAAATAACGGGAAGAGAA
Biotin-TEG (5’)
Capture
Probe
Aminated
CTAGAAATAACGGGAAGAGAA
Amine-C6 (5’)
Capture
6Fl~Q (3’)
Probe
(485/528 nm
excitation/emission)
Biotinylated CAAGATGTCCGCGTATTTAT
Biotin-TEG (3’)
Reporter
Probe
LiposomeCAAGATGTCCGCGTATTTAT
cholesteryl-TEG
imbedded
(3’)
Reporter
Probe
3.4 Labels
In order to determine the efficiency of probe immobilization and hybridization of
DNA target to the immobilized probes, fluorescently labeled probes and TAMRA labeled
target DNA were used. In the case of fluorescein, measurements were done using 485 and
528 nm for emission and excitation wavelengths. In the case of tetramethylrhodamine
(TAMRA), 540 nm and 590 nm were used for excitation and emission wavelengths.
13
3.5 Optimization of Streptavidin Liposomes
Liposome tagged with COOH were provided by Dr. Katie A. Edwards. Dr.
Edwards had originally developed a protocol for the coupling of streptavidin and other
proteins to the liposome surface using 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide·HCl (EDC) chemistry20. See Figure 5 for a
schematic of this chemistry. Here, the coupling protocol was optimized with respect to
liposome binding efficiency. Liposomes were characterized as follows.
COOH
+
CH3-N=C=N-(CH2)3-NH4+Cl(EDC)
COO
N-(CH2)3-NH4+
NH-CH2-CH3
+
NH2
CO-NH
+ H2N-CO-NH2 (Urea)
Figure 5. Schematic of EDC coupling chemistry. Carboxyl-tagged liposomes (purple)
bind EDC, and the resulting group will react with lysine residues in a protein (green),
coupling the protein to the liposome and producing urea as a byproduct.
The original protocol for coupling streptavidin to COOH-tagged liposomes was:
Add 100 µL of 0.1 M 2-(4-morpholino)-ethane sulfonic acid (MES) at pH 6.2 to 100 µL
of liposomes at the same time as the streptavidin is added at a concentration of 0.05
mol% of the total lipid concentration of the liposomes. Immediately following this, EDC
at pH 4.6 is added at 5 molar equivalents of EDC per mol of COOH-modified lipid in the
liposomes. This final solution is mixed vigorously on a vortex for 15 minutes, and then
14
separated in a column containing sepharose CL-4B beads (Sigma) in 1xHSS3.
Liposomes were characterized with respect to size, using dynamic light scattering (DLS)
with a DynaPro DLS laser (Protein Solutions), and phospholipid concentration, using the
Bartlett phosphate concentration determination assay2. Liposomes were also
characterized by encapsulation efficiency (i.e. ratio of dye concentration to total lipid
concentration) and binding efficiency.
For encapsulation efficiency, SRB was diluted serially in n-octyl-β-Dglucopyranoside (OG) to 0, 0.0001, 0.001, and 0.01 mg/mL, and 50 µL was used in
triplicate as a calibration curve in a 96-well plate. Conjugated liposomes were added at a
1:10 dilution in 30 mM OG for a final volume of 50 µL per well and fluorescence was
measured using the FLx800 Fluorescent Plate Reader (BIO-TEK Instruments, Inc.) at
excitation and emission wavelengths of 540 nm and 590 nm, respectively. All
measurements were done in triplicate unless otherwise specified. Binding efficiency was
measured using biotin-labeled plates (Pierce, Rockford, IL).
First, the plate was blocked by washing it once with 200 µL PBS and twice with
200 µL of a blocking solution (0.02 M Tris Base, 0.15 M sodium chloride, 0.01% sodium
azide, 0.3% gelatin and 0.02% Tween-20). The plate was then washed once with 200 µL
PBS and once with 200 µL HSS. 100 µL of the liposomes was added at between 0.01
and 0.1 mM total lipids and allowed to hybridize at room temperature in a drawer for 30
minutes. At this time, the plate was washed 3 times with 200 µL HSS and then 50 µL of
30 mM OG was added to lyse the liposomes. The plates were read at 540 nm excitation
and 590 nm emission wavelengths.
15
3.6 Evaluation and Comparison of Microtiter Plates
Five microtiter plates were used: NeutrAvidin Reacti-Bind plates (Pierce,
Rockford, IL), medium binding plate (Thermo, Milford, MA), high binding plate
(Greiner Bio-One, Monroe, NC) and two different amine binding plates, DNA-BIND
(Corning, Acton, MA) and Reacti-Bind Maleic Anhydride Plate (Pierce, Rockford, IL).
In all cases, the capture probe immobilization procedure was different. However, from
the target hybridization forward, the assay was the same as that typically used for the
NeutrAvidin plates (described below), unless otherwise noted, and all measurements
were done in triplicate.
3.7 Neutravidin-coated plates
NeutrAvidin Reacti-Bind plates were first washed twice with 200 µL of
phosphate buffered saline (PBS, 0.01 M potassium phosphate, 0.15 M sodium chloride,
0.01% sodium azide). Then, 100 µL of a 0.1 µM solution of biotinylated probe (capture
probe) in PBS was added into the wells and incubated for 30 minutes are room
temperature. The solution was then discarded and the plate washed twice more with 200
µL PBS and once with running buffer (30% v/v formamide, 1.35 M sodium chloride,
0.135 M sodium citrate, 0.05% sodium azide, 0.2% Ficoll type 400). Then, 100 µL of a
target sequence of DNA was added, diluted in running buffer to concentrations of 0, 0.1,
1, 5, 10, 50, 75, and 100 nM. The target was allowed to hybridize with the immobilized
capture probe for 30 minutes in a drawer. Subsequently, the remaining liquid was
aspirated and the plate washed twice with 200 µL running buffer and then once with 200
L running buffer with 0.2 M sucrose added. Then, 100 µL of liposomes tagged with the
reporter probe were added at 0.3 mM of phospholipids, diluted in running buffer-sucrose
16
and incubated for 30 minutes. The wells were washed three times with 200 µL HEPES
sucrose saline (HSS, 0.2 M sucrose, 0.2 M sodium chloride, 10 mM HEPES, 0.01%
sodium azide, pH 7). Finally, 50 µL of OG was added to lyse the liposomes. All plates
were read using Flx800 Fluorescent plate reader, with 540 nm and 590 nm excitation and
emission wavelengths. Figure 1a shows a cartoon of the complete assay.
3.8 Unlabeled plates
Medium and high binding unlabeled plates were first washed twice with 200 µL
PBS. The unlabeled capture probe was diluted to a concentration of 0.1 µM in a 1:1
solution of DNA Reacti-BIND immobilization solution (Pierce, Rockford, IL) and TE
(10 mM Tris-Cl, 1 mM EDTA, pH 9.2). This solution was gently rocked on a RotoShake Genie (Scientific Industries, Inc.) at a setting of 3 for 10 minutes at room
temperature before 100 µL were added to each well. The DNA was allowed to adsorb for
60 minutes at room temperature, during which the plate was covered with aluminum foil
and rocked gently on the Roto-Shake Genie at settling 1. Then the plate was washed
twice with 200 µL PBS and once with 200 µL of running buffer.
3.9 DNA-BIND plates
For the DNA-BIND plates, aminated capture probe (see table 1) was diluted to
0.1 µM in a solution of 500 mM Na2HPO4 and 1 mM EDTA (pH 8.5). 100 µL of this
solution was added to each well of the plate and incubated for 15 minutes at room
temperature. The wells are then washed three times with 200 µL of Tris-buffered saline
(TBS, 0.02 M Tris Base, 0.15 M sodium chloride, 0.01% sodium azide). 200 µL of a
block buffer (TBS with 0.05% Tween-20 and 0.1% casein) were then added to the wells
17
and incubated at room temperature for 15 minutes in a drawer. The block buffer was
removed and the plate washed once with 200 µL running buffer.
3.10 Reacti-Bind Maleic Anhydride plates
For the Reacti-Bind Maleic Anhydride plates, aminated capture probe (see table 1)
was diluted to 0.1 µM in PBS at pH 8.5. 100 µL of this solution was added to each well
of the plate, which was then covered with aluminum foil and rocked on the Roto-Shake
Genie at settling 1 for one hour at room temperature. After that, the remaining solution
was aspirated and 200 µL of a block buffer (TBS with 0.05% Tween-20 and 0.1% casein)
was added and allowed to incubate for one hour at room temperature, in a drawer. The
plate was then washed three times with 200 µL TBS with 0.05% Tween-20 and once with
200 µL running buffer. The OG and dye released by the lysed liposomes was transferred
to a black, unlabeled 96-well microtiter plate and then read at 540 nm and 590 nm
excitation and emission wavelengths.
3.11 Blocking Solutions
In order to optimize the immobilization and assay procedures for each plate, a
range of blocking buffer solutions were used. They are summarized in Table 2.
Experiments included the use of the different solutions, varying blocking incubation
times (1 – 20 min) and at various steps throughout the assay procedure. This included
blocking simultaneously with capture probe immobilization, directly after capture probe
immobilization, after target sequence addition, just prior to liposome addition and
simultaneous with liposome additions. In some cases, experiments were performed using
the fluorescently-labeled capture probe, the TAMRA labeled DNA target or the entire
assay including liposome binding. For both amine plates, blocking was done as per the
18
manufacturer’s protocol, and the NeutrAvidin plates are ordered pre-blocked. For
liposome optimization, blocking was carried out as described above.
Table 2. Blocking solutions and outline of investigation strategy. The blocking solutions
were used in plain styrene plates, a biotinylated plate, and a high-binding plate. Each
solution was tested in triplicate, arranged in a 96-well plate as indicated. Tests were
conducted in TBS, except for the high-binding plates which were done in TE.
Columns 1 – 3
Columns 4 – 6
Columns 7 – 9
Columns 10 – 12
Row
Unblocked
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
A
Row
0.1% BSA
0.1% BSA
0.1% BSA
0.1% BSA
B
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
Row
0.5% BSA
0.5% BSA
0.5% BSA
0.5% BSA
C
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
Row
1% BSA
1% BSA
1% BSA
1% BSA
D
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
Row
0.02% PVP
0.02% PVP
0.02% PVP
0.02% PVP
E
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
Row
0.2% PVP
0.2% PVP
0.2% PVP
0.2% PVP
F
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
Row
0.05% gelatin
0.05% gelatin
0.05% gelatin
0.05% gelatin
G
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
Row
0.5% gelatin
0.5% gelatin
0.5% gelatin
0.5% gelatin
H
0.02% Tween-20 0.05% Tween-20 0.1% Tween-20
3.12 Mixed assay format
The same procedure as described for the high binding plates was followed to
immobilize the capture probe DNA. Here, reporter probe-labeled liposomes were diluted
in running buffer with 0.2 M sucrose added to 0.05 mM phospholipids and mixed with
target ranging in concentration from 0 to 100 nM. This solution was incubated at room
temperature without shaking for 30 minutes, and then 100 µL of it was added to each
well and incubated again for 30 minutes at room temperature in a drawer. The plate was
then washed 3 times with 200 µL HSS, and then 50 µL 30 mM OG was added to each
19
well to lyse the liposomes. The plate was then read at 540 nm and 590 nm excitation and
emission wavelengths in FLx800 fluorescence reader.
3.13 Universal liposome assay
The same procedure as described for the Neutravidin-labeled plates was followed until
target sequence hybridization. Subsequently, the plate was washed twice with 200 µL
running buffer and then 100 µL of a biotinylated reporter probe was added, diluted to 0.1
µM in running buffer. This was allowed to hybridize for 30 minutes at room temperature
in a drawer, and then the plate was washed twice with 200 µL running buffer and once
with 200 µL HSS. Then, 100 µL of 0.3 mM phospholipid liposome solution was added
(diluted in HSS). The liposomes were tagged with 0.05 mol% of streptavidin. The
liposomes were incubated for 30 minutes in the wells at room temperature in a drawer.
Then the plate was washed three times with 200 µL HSS. Finally, 50 µL of 30 mM OG
were added and liposomes quantified at 540 nm and 590 nm excitation and emission
wavelength in FLx800 fluorescence reader.
20
Chapter 4– Results and Discussion
The two main objectives of the thesis were the optimization of an EDC-based
coupling protocol that couples streptavidin to COOH-labeled liposomes with respect to
liposome size, concentration, encapsulation efficiency and binding efficiency. The
second was to evaluate several different microtiter plates that use different DNA
immobilization strategies to determine their performance in a DNA and liposome-based
DNA detection assay. The performance of the plate was characterized both by the
magnitude of the overall signal and by the S:N.
4.1 Optimization of Streptavidin Liposomes
Streptavidin was coupled to liposomes following the previously optimized
protocol21. Liposomes were characterized using DLS and the Bartlett assay and were
determined to contain between 1 and 3 mM phospholipids and having an average
diameter of 292 nm +/- 33 nm. The original liposome solution, i.e. prior to coupling to
streptavidin, contained 15 mM phospholipids, thus, liposomes are diluted about 10 times
during the coupling procedure and subsequent column separation. The encapsulation
efficiency was about 0.6, similar to the uncoupled liposomes, and the binding efficiency
was deteremined to be 79,000 under optimal coupling conditions (pH 7) and at a total
lipid concentration of 0.1 mM (Figure 6, data plotted for pH 4, 6.2, and 7).
To optimize the performance of these liposomes, the volume and pH of MES
were varied between 0 and 200% of the liposome volume and pH 3.5 and 7, respectively.
Also, the pH of EDC was varied between 3.5 and 7, and the streptavidin concentration
was varied between 0.01 and 0.07 mol%. The reaction was allowed to run between 10
and 120 minutes. Surprisingly, it was found that only streptavidin concentration, MES
21
volume of 0% and a MES pH of 7 affected the liposomes’ characteristics. In the case of
0% MES buffer, the liposomes aggregated. DLS determinations showed an increase in
the average diameter from 227 +/- 32 nm to 361 +/- 85 nm. Changing the pH of the MES
buffer from originally 6.2 to pH 7 increased the performance of the liposomes (Figure 6).
Because the volume of MES used did not greatly effect the reaction (i.e.
variations of less than 10%), the amount was reduced to 50% of the liposome volume in
order to conserve reagents. However, the concentration of streptavidin was very
important to the binding efficiency of the liposomes (Figure 6). Increasing streptavidin
concentration resulted in increased binding ability and increased signal to noise ratios.
The difference between 0.05 mol% and 0.07 mol% under optimal conditions was only
14.5%. Thus, 0.05 mol% streptavidin was chosen as the final optimized conditions taking
cost of reagents and performance into consideration. Therefore, the optimal conditions
for this reaction were 0.1 M MES at pH 7 at 50% of the volume of the liposomes used,
along with EDC at pH 4.6 at 5 molar equivalents per mole of COOH (optimized
previously) on the liposomes and 0.05 mol% streptavidin. As incubation times had
limited effect on the overall reaction efficiency, an incubation time of as little as 15
minutes including occasional vortexing sufficed.
22
Unconjugated
pH 7
pH 6.2
pH 4
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
0.01
0.03
0.05
StAv 0.01%
0.1
0.01
0.03
0.05
0.1
0.01
StAv 0.03%
0.03
0.05
StAv 0.05%
0.1
0.01
0.03
0.05
0.1
StAv 0.07%
[TL] (mM)
Figure 6. Streptavidin liposome binding to a biotinylated plates. Variables included pH
of the MES buffer, streptavidin concentration (StAv %) and liposome concentrations
(0.01 – 0.1 mM total lipids). Liposomes were immobilized according to the procedure
described in chapter 3. After immobilization, liposomes were lysed in 30 mM OG and
signals were read at 540 nm excitation and 590 emission wavelengths. Analysis was
done using Microsoft Excel and standard deviations are represented as error bars.
In order to perform the binding assay shown in Figure 6, it was first necessary to
find an appropriate blocking procedure for the biotinylated plate used. The biotinylated
plates are polystyrene based, so blocking was first done in inexpensive, unlabeled black
styrene plates to determine the general composition of blocking solution needed. The
blocking procedure outlined in chapter 3 was used, and each of the solutions described in
Table 1 was tested. Unconjugated COOH-tagged liposomes were incubated in the
blocked plates for 30 minutes at room temperature without shaking in a drawer,
according to the procedure used, and non-specific binding was determined by reading the
23
plates in a fluorescent plate reader at 540 nm excitation and 590 nm emission
wavelengths. From these tests, it was found that gelatin-based blocking solutions were
the most effective. Here, background signals obtained were only about 5000
(fluorescence units) whereas all other conditions resulted in background signals between
5100 and 17500. Using these results, blocking solutions composed of TBS with a range
of gelatin (0 to 0.5%) and Tween-20 (0 – 0.1%) concentrations were tested in the
biotinylated plates, according to the blocking procedure outlined in chapter 3. It was
found that a blocking solution containing of 0.3% gelatin and 0.02% Tween-20 in 1xTBS
(0.02 M Tris Base, 0.15 M sodium chloride, 0.01% sodium azide) was the most effective
at reducing non-specific binding, and was used in all tests of binding efficiency for the
streptavidin liposomes. Non-specific binding was reduced from a signal of 19000 for
non-blocked plates, to about 5000 for non-optimized blocking solutions, which was also
the non-specific binding signal for the final optimized procedure.
24
Chapter 5 – Results and Discussion
5.1 Evaluation and Comparison of Microtiter Plates
Initially, all five microtiter plates with immobilized capture probes were
investigated using 0 to 100 nM of DNA target and 0.3 mM phospholipid reporter-probe
tagged liposomes as described in chapter 3. Here, capture probes were immobilized in all
cases following the manufacturer’s protocols. The general principle of probe
immobilization and liposome-assay is shown in figures 7 and 8 to indicate the difference
between the plates. Thus, in the case of NeutrAvidin plates, the capture probe is removed
from the plate surface and directed into the solution phase. In the case of adsorption, the
capture probe is oriented horizontally along the plate, attached at several points along the
backbone, with the bases directed into the solution phase. For covalent binding, the
capture probe is tethered to the plate by one end, and the length is directed into the
solution phase, similarly to the NeutrAvidin plates. Figure 7 shows the assay with DNAtagged liposomes, used to detect a specific target sequence, and Figure 8 shows a
universal assay using streptavidin-tagged liposomes and biotinylated probes.
25
NeutrAvidin
Adsorption
Covalent Binding
Legend
CP
Target
RP
Neutravidin
Biotin
Liposome
Figure 7. Cartoon depicting hybridization complexes for different microtiter plates. In
each case, capture probes are immobilized on a plate (grey) and hybridized with a target
DNA sequence which is hybridized to the reporter probe tagged to liposomes. Drawing is
not done to scale.
NeutrAvidin
Adsorption
Covalent Binding
Legend
CP
Target
RP
Neutravidin
Biotin
Streptavidin
Liposome
Figure 8. Cartoon depicting hybridization complexes for different microtiter plates and
streptavidin binding for biotinylated reporter probes. In each case, capture probes are
immobilized on a plate (grey) and hybridized with a target DNA sequence which is
26
hybridized to the biotinylated reporter probe that then binds to a streptavidin-tagged
liposome. Drawing is not done to scale.
Blocking of the plates was also performed according to these protocols. Surprisingly,
signals were only obtained from Neutravidin plates (Figure 9). All other plates did not
result in any positive signal. In order to elucidate the reason why the other plates did not
result in any detection of the target DNA, a number of experiments were performed. Here,
each step of the sandwich assay was investigated using fluorescein-labeled capture
probes and TAMRA labeled DNA target.
First, capture probes were immobilized on each plate and then hybridized with
TAMRA labeled DNA target. The resulting fluorescence was measured and plotted in
Figure 10. Except for the medium binding plate, all plates provided a dose response to the
varying concentrations of target sequence. Thus, at this point, the medium-binding plate
was discarded and not included in future experiments.
27
Neutravidin
Corning Amine Binding
Pierce Amine Binding
High Binding
70000
60000
Fluorescense
50000
40000
30000
20000
10000
0
0
20
40
60
80
100
120
Target Concentration (nM)
Figure 9. Comparison of different microtiter plates in a liposome-enhanced DNA
sandwich assay. In all cases, capture probes were immobilized using manufacturer’s
protocols, target DNA was allowed to hybridize and finally reporter probe-tagged
liposomes encapsulating SRB were bound. The high binding plate was unblocked. Upon
liposome lysis using 30 mM OG, signals were measured at 540 nm and 590 nm excitation
and emission wavelengths, respectively. Analyses were done in Microsoft Excel, and
standard deviations were plotted as error bars.
28
High Binding
Neutravidin
Medium Binding
Corning Amine Binding
4500
4000
3500
Fluorescense
3000
2500
2000
1500
1000
500
0
0
20
40
60
80
100
120
Target Concentration (nM)
Figure 10. Comparison of different microtiter plates at the target hybridization step in a
liposome-enhanced DNA sandwich assay. In all cases, capture probes were immobilized
using manufacturer’s protocols, and TAMRA-labeled target was allowed to hybridize.
The high binding and medium binding plates were unblocked. The TAMRA
fluorescence was read in 100 µL running buffer at 540 nm and 590 nm excitation and
emission wavelengths, respectively. Analyses were done in Microsoft Excel, and
standard deviations were plotted as error bars.
In order to further investigate the fact that no liposome binding was observed in
all plates except for the NeutrAvidin coated plates, several experiments were designed
using the high binding plates to determine whether increasing the length of the liposomebound probe would facilitate binding. These experiments included using a reporter probe
with a C18 spacer between its 3’end and the liposome, or pre-hybridizing the target with
the DNA-tagged liposomes and then binding this complex to the capture probe (Figure 12,
“sequential”), or some combination of the two (see Figures 7 and 8 for sequential assays
and Figure 11 for mixed assay schematic).
29
Legend
CP
Target
RP
Time
Running
Buffer
Liposome
Figure 11. Cartoon depicting pre-hybridization assay format using adsorption capture
probe immobilization. The target and DNA-tagged liposomes are hybridized first,
separate from the plate. Then, the liposome-DNA probe-target complex is added to a
plate (grey) that already has capture probe immobilized and is allowed to hybridize.
Drawing is not done to scale.
At the same time, two additional variations were designed to determine (1) if the capture
probe/target complex was washing off when the liposomes were added binding target
probe directly to the plates (Figure 12, “target immobilized”) instead of hybridization to a
capture probe. (2) Using DNA-tagged liposomes or streptavidinylated liposomes and
biotinylated reporter probes, to determine if the protein, which is much larger than a
DNA probe, would provide enough space to allow the liposomes to bind to the reporter
probe. As seen in Figure 9, in only one condition, positive signals were obtained. Only
the streptavidin-tagged liposomes produced a dose-response over the target range of 0 to
100 nM. In contrast, for the experiment in which target was bound directly to the plate
and then a biotinylated reporter probe and streptavidin-tagged liposomes were used, there
is a very low and erratic signal over the target concentration range. This indicates that,
the liposomes have a minimum distance from the plates which they must maintain, and
30
which is very close to the distance achieved in the streptavidin liposome assay in which
capture probe is bound to the plate. So it is likely that the other formats tested failed
because the liposomes were unable to approach the plate close enough to allow the
reporter probe, or target/reporter probe complex, to hybridize to the DNA already on the
plate.
12000
10000
8000
6000
4000
2000
Sequential
Sequential
DNA-tagged DNA-tagged
Liposomes Liposomes,
Target
immobilized
Sequential
StAv
Liposomes
50
100
0
10
50
100
0
10
50
100
10
0
50
100
0
10
50
100
0
10
50
100
0
10
50
100
0
10
50
100
0
10
0
Sequential Mixed Target Mixed target Sequential
Sequential
StAv
and DNAand DNADNA-tagged DNA-tagged
Liposomes,
tagged
tagged
Liposomes
Liposomes
Target
Liposomes
liposomes
with C18
with C18
immobilized
with C18
spacer
spacer,
Assay Format and [Target] (nM)
Figure 12. Comparison of assay formats for liposome-enhanced sandwich assay to
determine viable procedures. For each assay, capture probe or target DNA was
immobilized on a high binding plate according to the protocol described in chapter 3, and
so in all assays, the plates were unblocked. Then, either a sequential format or a mixed
format was followed. The sequential format consists of hybridizing target to
immobilized capture probe, and then either DNA-tagged liposomes to this complex or a
biotin-labeled reporter probe followed by streptavidin liposomes. The mixed format
involved mixing the target and DNA-labeled liposomes and then adding this mixture to
wells already coated with capture probe. Liposomes were added at a concentration of
0.05 mM phospholipids and lysed with 50 µL of 30 mM OG. Plates were then read at
540 nm and 590 nm excitation and emission wavelengths, and data was analyzed in
Microsoft Excel. Standard deviations are plotted as error bars.
31
Based on these findings, streptavidin-labeled liposomes were used in each type of
plate. The manufacturer’s protocols were followed to immobilize 0.1 µM of capture
probe on the high binding, NeutrAvidin, Pierce amine-binding, and Corning aminebinding plates, followed by the hybridization of target DNA over a range of 0 – 100 nM,
0.1 µM of reporter probe, and 0.3 mM phospholipid streptavidin liposomes according to
the procedure described in chapter 3 under “Universal Assay.” The results of these
experiments are shown in Figure 13. As was expected, for each plate, a positive signal
resulted, with the strongest signals generated by the NeutrAvidin plates. The results
obtained here followed results shown in Figure 10, indicating signals did not depended on
the amount of target captured in each plate. While the maximum signal from the
NeutrAvidin plates is about more than twice that of the high binding plate’s, the signal to
background noise ratio is much lower, at a maximum of about 1.5:1 compared to 78:1 for
the high binding plates. This indicates that despite blocking, the non-specific binding on
the NeutrAvidin is much higher than the non-specific binding occurring for the high
binding plate. This may be occurring because both streptavidin and neutravidin are used
in this assay, and both proteins bind biotin which is used twice in this assay, and so the
immobilized capture probe may be interfering with the ability of the liposomes to bind
the reporter probe.
32
Corning Amine Binding
NeutrAvidin
Pierce Amine Binding
High Binding
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
0
20
40
60
80
100
120
-5000
[Target] (nM)
Figure 13. Comparison of different microtiter plates in a universal liposome-enhanced
DNA sandwich assay. In all cases, capture probes were immobilized using
manufacturer’s protocols, target DNA was allowed to hybridize, followed by a reporter
probe which was also allowed to hybridize, and finally streptavidin-tagged liposomes
encapsulating SRB were bound. The high binding plate was unblocked. Upon liposome
lysis using 30 mM OG, signals were measured at 540 nm and 590 nm excitation and
emission wavelengths, respectively. Analyses were done in Microsoft Excel, and
standard deviations were plotted as error bars.
The manufacturer’s protocol for the use of DNA Reacti-bind immobilization
solution in enhancing the adsorption of DNA to the high binding (and medium binding)
plates was very general, and so some optimization of the amount of capture probe
adsorbed was done to achieve the results shown in Figures 9, 10, 12, and 13. The
immobilization was optimized with respect to the diluent used in the immobilization
solution and the percentage of the solution the diluent makes up, the pH of this solution,
the capture probe concentration needed, and how long the solution should be incubated in
33
the plate with the capture probe to achieve maximal probe adsorption. TE, TBS,
deionized water, and PBS were tested as diluents and a range of 10% to 66.7% diluent
was tested. Also, a solution pH range of 5.2 to 9 was tested for the best of these solutions,
along with a time range of one to 24 hours and a capture probe concentration range of 0
to 0.15 µM. It was found that the best solution was DNA-Reacti-bind diluted with 50%
TE at pH 9, yielding a final solution pH of 7.1. This solution works most effectively
when combined with 0.1 µM capture probe and incubated in the plate for 1 hour, as
described in chapter 3. Also, the manufacturer does not recommend a specific blocking
procedure. All of the blocking solutions listed in Table 1 were tested, with blocking
occurring before, during, or after capture probe immobilization or target hybridization, as
described in chapter 3. BSA-based blocking solutions seemed the most effective at
reducing non-specific binding, but all permutations interfered with DNA immobilization
or hybridization enough to cancel out the positive effect of reducing the background
noise. Therefore, no blocking was used in the assays which generated the data shown in
Figures 9 - 13.
The universal assay using streptavidin-tagged liposomes was also optimized using
the high binding plates. A range of reporter probe (0.05 – 1.0 µM) and streptavidintagged liposomes (0.05 – 0.8 mM phospholipid) were tested concurrently at target
concentrations of 0 and 50 nM. 50 nM target was chosen because this is the
concentration that yields the peak signal and signal to noise ratio in these assays. It was
found that 0.1 µM reporter probe and 0.2 mM phospholipid produced the highest signal
to noise ratio (~60:1) while maintaining a high signal. These is very similar to the
sequential assay used with the NeutrAvidin plates and reporter probe-tagged liposomes,
34
which have a 0.13 µM reporter probe concentration and 0.2 – 0.3 mM phospholipid
liposome concentration for the best performance.
35
Chapter 6 – Conclusions and Future Work
6.1 Optimization of Streptavidin Liposomes
The results of the streptavidin liposome optimization show that the EDC-mediated
fast coupling reaction was most efficient at a pH of 7 and a streptavidin concentration of
0.05 mol%. The buffer, 0.1 M MES at pH 7, was added at 50% of the volume of
liposomes used, and the EDC was at a concentration of 5 molar equivalents per mole
COOH. The reaction worked best following the original protocol for mixing the reagents,
and requires at least 15 minutes of vortexing.
During this optimization, it was noticed that the coupled liposomes had slightly
lower encapsulation efficiencies than the uncoupled COOH-tagged liposomes, indicating
there is SRB leakage during the process. It was found that the EDC contributes to this
leakage, and it was also noticed that the liposomes swell from 213 nm +/- 39 nm in
diameter (the diameter of the uncoupled liposomes) to 292 nm +/- 33 nm, which may also
account for some of the leakage. Further investigation could be done into limiting the
effect EDC has on encapsulation efficiency and reducing the liposome swelling. This
might be accomplished by adding 0.2 M sucrose to the MES to equalize the osmolarity of
the MES with the SRB encapsulated in the liposomes. Additionally, the amount of
streptavidin actually coupling to the liposomes is unknown, so an investigation into how
much streptavidin is on the liposomes would be beneficial both for further characterizing
the liposomes and also for improving the universal assay format that uses these liposomes.
This could be done either by creating liposomes containing only buffer and no dye and
coupling them to fluorescently labeled streptavidin. Using this method, the phospholipid
concentration can still be determined, and then the amount of streptavidin per mol of
36
phospholipid can be determined by creating a streptavidin standard in a microtiter plate
and measuring the fluorescence from the bound streptavidin. This fluorescence would
possibly be increased by lysing the liposomes first, so measurements should be taken
before and after. Another possible method would be to use one of the many protein
assays commonly available22 to quantify the amount of streptavidin, again, relative to the
phospholipid concentration. There is a possibility that the phospholipids themselves or
the encapsulated dye would interfere with these assays and so any potentially useful
assays must be chosen carefully.
6.2 Evaluation and Comparison of Microtiter Plates
For high-throughput DNA sandwich assays, as described in chapter xxx, it was
found that NeutrAvidin-labeled plates performed the best, with a signal to noise ratio of
over 400:1 for 50 nM of target sequence These plates work best when a DNA-tagged
liposome is used, and thus are the best choice for a specific assay. For a more universal
assay which uses streptavidin-tagged liposomes and biotinylated reporter probes, a high
binding plate in conjunction with DNA Reacti-bind provided the best results with a signal
to noise ratio of about 80:1 for 50 nM of target sequence. Amine-binding plates did not
perform well in either the specific or universal assay and are not recommended. Medium
binding plates are also not recommended, as they exhibited high variability and poor
repeatability in these experiments.
The most surprising result of this study was that only the NeutrAvidin plates will
work for a DNA sandwich assay using DNA-tagged liposomes. It was suspected that this
was due to the increased space between the plate and liposomes provided by the
NeutrAvidin, which is approximately 6 nm thick, compared to 1 nm or less in separation
37
between the capture probe and plate in the other immobilization methods. More
investigation could be done to verify that this happened because the liposomes cannot
approach the plates closely enough to allow hybridization to occur. Also, determining
the chemical and physical properties of this assay that prevent the liposomes from getting
close enough to the plates may be useful, if this is the reason for the failure of most of the
plates. With this information, improvements might be made to the assay to obtain
positive results for the high binding or amine plates. Additionally, more work could be
done on blocking both the NeutrAvidin and high binding plates to improve their
performance in the universal assay. Some blocking was attempted for the high binding
plate, but proved to be difficult because it interferes with capture probe immobilization
and DNA hybridization. Blocking reagents that might be useful are 3% BSA, 0.2 M NaCl,
0.1 M Tris–HCl (pH 8.0), and 0.05% Triton X-100) or 1 mg/mL BSA made up in
1 × phosphate buffer saline, because they have been used previously by Kimura and Lillis,
respectively, in DNA adsorption and hybridization assays23,24.
38
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