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Electrochemical Based Detection of Influenza
by
Xiao Guo
A thesis submitted in conformity with the requirements for the degree of Master of Science
Biochemistry University of Toronto
© Copyright by Xiao Guo 2013
ii
Electrochemical Based Detection of Influenza
Xiao Guo
Master of Science
Biochemistry
University of Toronto
2013
Abstract
Influenza is the infectious agent of the seasonal flu. Flu symptoms from influenza infection are
similar to the symptoms caused by bacterial upper respiratory tract infections. This similarity
causes the inappropriate diagnosis and prescription of antibiotics, leading to drug resistant
bacterial strains. Moreover, the limitations of the current viral detection methods prevent the
clinical diagnosis of influenza.
The objective of this project is to design a rapid and sensitive influenza diagnostic method based
on the highly sensitive Nanostructured microelectrode biosensing assay.
The diagnostic method was designed by selecting probe sequences, controlling the quality of the
probes and the sensing chips, and optimizing the deposition conditions. This diagnostic method
was shown to be capable of differentiating influenza sequences from non-complementary
sequences, detecting influenza sequences in the form of ~1000-nucleotide RNA molecules,
sensing the target influenza RNA within a complex mixture of cell lysates, and achieving a
clinically relevant detection limit.
iii
Table of Contents
Table of Contents ........................................................................................................................... iii
List of Tables ................................................................................................................................. vi
List of Figures ............................................................................................................................... vii
List of Abbreviations ..................................................................................................................... ix
List of Appendices ......................................................................................................................... xi
Chapter 1 Introduction .................................................................................................................... 1
1 Project Rationale ........................................................................................................................ 1
1.1 Motivation ........................................................................................................................... 1
1.2 Objectives ........................................................................................................................... 2
2 Literature Review ....................................................................................................................... 2
2.1 Influenza Virus and Antimicrobial Resistance ................................................................... 2
2.2 Current Influenza Detection Assays ................................................................................... 4
2.3 NME Biosensing Assay ...................................................................................................... 7
Chapter 2 Influenza Sequence Design and Assay Optimization .................................................. 12
3 Overview .................................................................................................................................. 12
4 Materials and Methods ............................................................................................................. 12
4.1 Materials ........................................................................................................................... 12
4.2 Probe Sequence Design ..................................................................................................... 14
4.3 Probe Synthesis and Quality Control: PNA Synthesis ..................................................... 15
4.4 Probe Synthesis and Quality Control: HPLC Purification and MS Analysis ................... 16
4.5 Probe Synthesis and Quality Control: Gel Binding Assay ............................................... 17
4.6 Chip Cleaning and Quality Control: Chip Cleaning ......................................................... 17
4.7 Chip Cleaning and Quality Control: Acid Scan ................................................................ 18
4.8 NME Formation: Microelectrode Formation .................................................................... 18
iv
4.9 NME Formation: Nano Structure Formation .................................................................... 18
4.10 NME Biosensing Assay: Probe Deposition ...................................................................... 18
4.11 NME Biosensing Assay: Pre Hybridization Scan ............................................................. 19
4.12 NME Biosensing Assay: Hybridization ............................................................................ 19
4.13 NME Biosensing Assay: Post Hybridization Scan ........................................................... 19
4.14 Probe Deposition Condition Optimization ........................................................................ 21
5 Results and Discussion ............................................................................................................. 21
5.1 Probe Sequence Selection ................................................................................................. 21
5.2 Probe Quality Control ....................................................................................................... 23
5.3 Chip Quality Control ......................................................................................................... 27
5.4 Probe Deposition Condition Optimization ........................................................................ 28
Chapter 3 Influenza Biosensing .................................................................................................... 31
6 Overview .................................................................................................................................. 31
7 Materials and Methods ............................................................................................................. 31
7.1 Materials ........................................................................................................................... 31
7.2 Target Vector Design ........................................................................................................ 32
7.3 In vitro Transcription ........................................................................................................ 32
7.4 Cell Transfection ............................................................................................................... 33
7.5 Cell Lysis and Lysate Preparation .................................................................................... 34
7.6 QPCR Analysis ................................................................................................................. 34
8 Results and Discussion ............................................................................................................. 34
8.1 Target Vector Design ........................................................................................................ 34
8.2 DNA Target Biosensing .................................................................................................... 36
8.3 In vitro Transcribed RNA Target Biosensing ................................................................... 39
8.4 Cell Lysate Biosensing ..................................................................................................... 41
8.5 RNA Quantitation ............................................................................................................. 43
v
Chapter 4 Conclusion and Future Directions ................................................................................ 45
9 Conclusion................................................................................................................................ 45
10 Future Directions ...................................................................................................................... 45
References ..................................................................................................................................... 48
Appendix I: Mass Spectrometry Spectrum ................................................................................... 50
vi
List of Tables
Table 1: The current assays for diagnosing influenza infection. .................................................... 5
Table 2: The components of the NME biosensing device. ............................................................. 9
Table 3: The properties of the selected probe sequences. ............................................................. 23
vii
List of Figures
Figure 1: The influenza virion. ....................................................................................................... 3
Figure 2: The procedure of the NME biosensing assay. ................................................................. 8
Figure 3: The structure of a nanostructured microelectrode (NME). ........................................... 10
Figure 4: The top and cross-section of the NME biosensing chip. ............................................... 10
Figure 5: The sensitivities and capabilities of the NME biosensor. ............................................. 11
Figure 6: The procedure for designing the probe. ......................................................................... 14
Figure 7: The procedure for synthesizing and characterizing the probe. ...................................... 16
Figure 8: The procedure for characterizing and cleaning the chip. .............................................. 17
Figure 9: A Differential Pulse Voltammetry (DPV) plot used to assay for target hybridization. 20
Figure 10: The acetonitrile gradient of the HPLC methods used to purify the probes. ................ 24
Figure 11: The HPLC chromatograms showing the purification of the probes. ........................... 25
Figure 12: The gel binding assay confirming the specific hybridization of the PNA probes to the
target DNA. ................................................................................................................................... 26
Figure 13: The acid scan checking the gold leads for defects and contaminants. ........................ 28
Figure 14: The NME biosensing assay comparing different deposition conditions. .................... 29
Figure 15: The plasmids for generating the influenza RNA sequences. ....................................... 35
Figure 16: Representative DPV scans using the NME biosensing assay. .................................... 36
Figure 17: The detection of DNA targets with the NME biosensing assay. ................................. 38
Figure 18: The detection of RNA targets with the NME biosensing assay. ................................. 40
viii
Figure 19: The detection of influenza RNA targets from cell lysates using the NME biosensing
assay. ............................................................................................................................................. 42
Figure 20: The determination of influenza RNA concentration from lysate using QPCR. .......... 44
ix
List of Abbreviations
CV Cyclic Voltammetry
DCM Dichloromethane
DCPA Direct Current Potential Amperometry
DMF N,N-Dimethylformamide
DPV Differential Pulse Voltammetry
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
FBS Fetal Bovine Serum
HATU 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate
Methanaminium
HPLC High Pressure Liquid Chromatography
MC Microfabricated Cantilevers
MCH Mercaptohexanol
MEM Minimal essential medium
MS Mass Spectrometry
NME Nanostructured Microelectrode
NMM N-Methylmorpholine
PBS Phosphate Buffer Saline
PNA Peptide Nucleic Acid
QCM Quartz Crystal Microbalance
x
QPCR Real time PCR
RTPCR Reverse Transcriptase Polymerase Chain Reaction
SPR Surface Plasmon Resonance
TBE Tris Borate EDTA
TFA Trifluoroacetic Acid
TIPS Triisopropyl silane
xi
List of Appendices
Appendix I: Mass Spectrometry Spectrum 50
1
Chapter 1 Introduction
1 Project Rationale
1.1 Motivation
Influenza is a virus that infects the human respiratory tract and is responsible for the seasonal
flu1. Unlike the viruses that are responsible for the common cold, influenza is highly pathogenic
and may lead to fatal pneumonia1. Influenza is also highly contagious and capable of escaping
the human immune defenses, resulting in seasonal epidemics and global pandemics2. In addition,
inappropriate prescription of antibiotics to the large number of patients suffering from influenza
infections results in the emergence of drug resistant bacterial strains3. Therefore, there is a need
for rapid and sensitive biosensing techniques to diagnose influenza.
Currently, there are 4 main types of diagnostic methods for detecting influenza infection.
However, their limitations make them unsuitable for usage in a clinical setting. Cell culture is
sensitive and specific, but it is very slow and requires trained technicians4. Direct Fluorescent
Antibody Staining is faster than cell culture, but it requires specialized instruments and trained
technicians4. Rapid antigen immunoassays is very fast and simple to perform, but it has very
poor sensitivity4. RTPCR is highly sensitive and specific, but it requires trained technicians and
expensive instruments4.
The Kelley group has developed a biosensing assay based on the hybridization of nucleic acids
and the measurement of electrochemical signals5. This assay is named the nanostructured
microelectrode (NME) biosensing assay, based on the type of electrode used to measure the
signals5. This technique capitalizes on the highly specific and relatively stable base-pairing
interactions to capture specific target sequences5. The hybridization event leads to the generation
of an electrochemical signal, which is very rapid and inexpensive to analyze5. The signal is
amplified and the background is minimized by using nanostructured microelectrodes, peptide
nucleic acid (PNA), and a ferricyanide and ruthenium hexamine reporter system to achieve very
high sensitivity6. A diagnostic device suitable for the clinical setting can be constructed by
modifying this biosensing technique to detect influenza viral sequences.
2
1.2 Objectives
The overall goal of the project is to create a biosensing device that is suitable for diagnosing
influenza infections in the clinical setting. There are 2 specific objectives. The first objective is
to design and modify the components of the NME biosensing assay to optimize the detection of
influenza sequences. The second objective is to test the ability of the assay to detect influenza
sequences in the forms of DNA oligonucleotides, in vitro transcribed RNA, and unpurified RNA
from transfected cell lines.
2 Literature Review
2.1 Influenza Virus and Antimicrobial Resistance
Influenza virus is a member of the Orthomyxoviridae family of viruses2. It is classified into A, B,
and C types based on 2 conserved proteins: nucleoprotein and matrix protein2. Influenza A is
further classified into 16 H subtypes (1-16) and 9 N subtypes (1-9) based on the membrane
glycoproteins: hemagglutinin and neuraminidase7. The most common influenza A subtypes are
H1N1, H2N2, and H3N27. Influenza B has only one common subtype
7. Influenza C is mainly
associated with mild cold-like illnesses7.
The influenza viral particle is spherical with an average diameter of 100nm, consisting of RNA
molecules, proteins, and lipids (Figure 1)8. The influenza genome is composed of 8 segments of
linear single stranded negative-sense RNA molecules (Figure 1)8. The viral genome is associated
with RNA binding proteins, which are housed within a lipid bilayer envelope (Figure 1)9.
Outbreaks that lead to seasonal epidemics and global pandemics are mainly caused by influenza
A and B, which affect 20% of children and 5% of adults worldwide1. This high frequency is
largely due to the high rate of viral antigenic change, which generates new viral strains that the
human population has no immunity to10
. Seasonal epidemics occur annually in the winter
months11
. Children, elderly, pregnant women, and chronically ill patients are at high risk for the
infection11
. Each year, influenza infections account for more than 20 000 deaths in North
America4. Pandemics have a large death toll, affect all age groups, and occur on a global scale
every 10 to 50 years4. For instance, the Spanish Flu caused 50 million deaths, and the Asian Flu
and Hong Kong Flu caused 1 to 2 million deaths each12
. Influenza infections therefore affect a
3
large population globally, and have a large toll on human welfare, economy, and health care
system4.
Figure 1: The influenza virion.
The spherical influenza particle is composed of a lipid bilayer envelope, which houses the
genome and the viral proteins. Influenza genome has 8 segments of linear single stranded
negative-sense RNA molecules, which are associated with the nucleoproteins. The matrix
proteins associate with the envelope. Hemagglutinin and neuraminidase are viral receptors
located on the surface of the envelope.
The influenza virus is highly contagious and can cause serious illnesses2. It transmits rapidly in
the population via respiratory aerosols and contaminated surfaces13
. The virus infects and kills
lung epithelial cells in the upper respiratory tract, leading to inflammation14
. This results in the
acute disease, displaying the symptoms of high fever, muscular pain, headache, cough, sore
throat, and nasal congestion15
. In certain cases, the loss of the lung epithelium allows bacteria to
colonize the lower respiratory tract and cause fatal pneumonia16
.
Three quarters of all prescribed antibiotics are used to treat respiratory infections, even though
the majority of respiratory tract infections are caused by viruses including influenza3. Antibiotics
target bacterial factors, and therefore have no effect on the progression of viral diseases like
influenza infection3. However, antibiotics apply a selection pressure on the bacteria present in
the patient and enrich the drug resistant bacterial strains3. The 3 major bacterial species that
Lipid bilayer envelope
RNA genome
Matrix protein
Nucleoprotein
Neuraminidase
Hemagglutinin
4
cause secondary infections in flu patients (Streptococcus pneumoniae, Haemophilus influenzae,
and Staphylococcus aureus) are all already resistant to penicillin and macrolid classes of
antibiotics17
. One study showed that the emergence of antibiotic resistant infections increased the
duration of hospital stay, mortality, morbidity, and treatment costs by two folds17
. Another study
found that nearly 80% of the flu patients that received antibiotics were not suffering from any
secondary bacterial infection3. These inappropriate prescriptions cost the United States
approximately $211 million annually3. These studies showed that the inappropriate prescription
of antibiotics not only decreases the effectiveness of the antibiotics, but it also increases health
care costs.
2.2 Current Influenza Detection Assays
The major barrier in reducing inappropriate prescription of antibiotics is a lack of reliable clinical
diagnosis of influenza infections, since bacterial and viral respiratory infections produce very
similar clinical pathologies17
. Often, suspected flu patients are given medication without
diagnosis17
. Currently, the assays used to detect influenza are based on viral culture, or antigen
recognition, or PCR. When diagnostic tests are performed on respiratory infection patients, the
majority of diagnostic samples are collected in the form of bronchial alveolar lavage18
. However,
each of these methods has limitations that make them unsuitable for clinical diagnosis.
The historically earliest influenza diagnosis is growing the virus in the conventional cell culture
or the Shell Vial Cell Culture (Table 1)19
. In the conventional cell culture, the viral specimen is
inoculated into a permissive cell line, such as the Madin Darby Canine Kidney cell19
. After
incubation, the infected cells display cytopathic effects, including rounding and degeneration20
.
In the Shell Vial Cell Culture, the virus is inoculated into a cell culture grown in a shell culture
tube, and then centrifuged at low speed to promote the entry of the virus into the cell20
. After a
short incubation time, the cell culture displays cytopathic effects20
. The Shell Vial Cell Culture
shortens the time required for the virus to infect the cell16
. The advantages of the culture
techniques are high sensitivity and generation of viable virus for further testing (Table 1)20
.
However, trained technicians are needed, and the time requirements (10 days for conventional
culture and 2 days for shell vial cell culture) are too long to affect patient management (Table
1)20
.
5
Table 1: The current assays for diagnosing influenza infection.
The sensitivity, specificity, time required, equipment required, technical expertise required,
cost, and throughput of the assays are compared. The sensitivity and specificity are
standardized as a percentage relative to the conventional cell culture, which is considered
as the gold standard assay. The cost refers to the money required to assay a single sample.
Low throughput refers to analyzing 1 sample at a time, medium throughput refers to
analyzing up to 10 samples, and high throughput refers to analyzing up to 100 samples.
Cell culture based assays have high sensitivity and selectivity, but are slow and require
technical expertise. Direct Fluorescent Antibody assay is relatively fast and inexpensive,
but requires expensive instruments. Rapid Antigen Immunoassay is very fast and simple to
use, but has low sensitivity. PCR based assays have high sensitivity and selectivity, but are
very expensive.
The Direct Fluorescent Antibody (DFA) Staining and Rapid Antigen Immunoassay techniques
are based on antigen recognition (Table 1)21
. In the DFA assay, fluorophore-conjugated
antibodies specific for influenza surface antigens are applied to influenza infected cells on a
slide21
. The labeled antibodies bind to the influenza particles, which are visualized with a
fluorescent microscope4. This assay takes less time to perform than the cell culture assays (Table
1)4. However, DFA assay requires trained technicians and expensive instruments, and it has
lower sensitivity compare to the cell culture assays (Table 1)4. In the rapid antigen immunoassay,
antibody 1 is bound to a solid support and targets an influenza antigen, while antibody 2 is
labeled with a chromogenic indicator and targets a different influenza antigen16
. Antibody 2
associates with the viral particle when incubated together in a buffer16
. Antibody 1 captures the
complex of antibody 2 and viral particle when the buffer passes over the solid support16
. The
6
result is the deposition of the chromogenic indicator at the location of antibody 1, which is
visualized by the naked eye16
. Although the rapid antigen immunoassay is simple and quick to
perform, its sensitivity is more than 1000 times lower than the cell culture methods (Table 1)4.
The reverse transcriptase PCR (RTPCR) is a PCR based diagnostic assay (Table 1). In this
method, the viral genomic RNA is converted to cDNA by reverse transcriptase21
. Specific
primers and DNA polymerase are used to copy the cDNA multiple times through several cycles
of amplification21
. The PCR can be automated and multiplexed to analyze multiple samples in
high throughput (Table 1)21
. Moreover, this method is much more sensitive than all the previous
detection methods, capable of detecting 5 to 10 viral particles (Table 1)21
. However, it requires
expensive equipment, reagents, and trained technicians (Table 1)4.
An emerging technique in disease diagnostics detects the hybridization of a known nucleic acid
probe sequence with a nucleic acid target sequence from the sample5. Hybridization is based on
the specific and relatively stable base-pairing interactions between complementary nucleic acid
molecules5. In this assay, a nucleic acid probe sequence is first immobilized on a surface
20. Then,
the complementary target nucleic acid sequence binds to the probe sequence20
. The hybridization
event is then measured optically, mechanically, or electrochemically, depending on the sensing
device20
.
Microarray and surface plasmon resonance (SPR) use optical readouts to detect hybridization,
while quartz crystal microbalance (QCM) and microfabricated cantilevers (MC) use mechanical
readouts to detect hybridization20
. The advantages of these methods are their high sensitivities20
.
However, the assays based on optical and mechanical readouts all have technical limitations,
from the degradation and aggregation of fluorescent labels for microarray, to the requirement of
massive quantities of probe deposition for SPR, to the difficulties of assaying in liquid phase for
QCM, to the challenges of fabricating cantilever features for MC20
. In addition, all of the above
hybridization based methods require expensive equipment and sophisticated numerical
algorithms, which are better suited in a laboratory rather than a clinic20
. Fortunately,
hybridization methods that use electrochemical readouts combine the advantage of high
sensitivity with inexpensive device and simple data analysis20
.
There are three diagnostic methods that measure an electrochemical signal. In the
Electrochemistry of Nucleic Acid method, the electrode oxidizes the guanine bases on the
7
hybridized sequences and measures the current from the redox reaction5. This method is label
free, but its sensitivity depends on the amount of guanine present in the sequence5. In the Nucleic
Acid Specific Redox Indicator method, the target sequence is labeled with a redox active reporter
such as a metal nanoparticle5. The reporter on the target sequence generates a characteristic
electrochemical signal that is measured by the electrode5. This method is very sensitive, but it
requires sample labeling5.
In the Nucleic Acid Mediated Charge Transport Electrochemistry method, a reporter molecule
such as ruthenium hexamine is recruited to the hybridized double stranded nucleic acid and
undergoes a measurable redox reaction5. This method combines the advantages of the other
electrochemical based hybridization assays5. It is label free, simple, robust, and requires only one
hybridization event5. The sensitivity of this method can be further improved by modifying the
reporter molecule, probe molecule, and electrode structure.
2.3 NME Biosensing Assay
The NME biosensing assay improves upon the Nucleic Acid Mediated Charge Transport
Electrochemistry method. This technique has 5 major steps. The first step is forming the
nanostructured microelectrode (NME) via electroplating (Figure 2)22
. The NME is a working
electrode that is ~30µm in size and contains nanometer scale features (Figure 3)22
. The second
step is attaching the peptide nucleic acid (PNA) probes onto the NME (Figure 2)22
. PNA is a type
of biological polymer that can hybridize to nucleic acids via base-pairing22
. The third step is
scanning the probe modified NME with ferricyanide and ruthenium hexamine redox reporters to
determine the background signal (Figure 2)22
. The next step is hybridizing the target nucleic acid
to the probes (Figure 2)22
. The final step is scanning the NME with the redox reporters to
measure the signal after target hybridization (Figure 2)22
. Each component of the NME
biosensing assay (electrochemical measurement, nucleic acid hybridization, ferricyanide and
ruthenium hexamine redox reporter, PNA, and NME) contributes to improve the sensitivity and
selectivity of the assay (Table 2).
8
Figure 2: The procedure of the NME biosensing assay.
A) First, gold and palladium are electroplated onto the lead to form the nanostructured
microelectrode (NME). Gold plating generates the microelectrode, while palladium plating
generates the nanostructures on the electrode. B) Next, the peptide nucleic acid (PNA)
probe is attached onto the NME. The PNA probe is a biological polymer composed of a
charge neutral peptide back bone and nucleic acid bases, which can hybridize to nucleic
acid molecules. C) Then, the background current from the redox reaction of the ruthenium
hexamine and ferricyanide electrochemical reporters is scanned with differential pulse
voltammetry (DPV). Ruthenium hexamine is reduced by the electrode, while ferricyanide
regenerates the oxidized form of ruthenium hexamine. D) After that, the sample DNA or
RNA target sequence is hybridized onto the probe. E) Finally, the electrochemical current
is measured after hybridization, to determine the signal increase from the redox reporters
that are recruited by the target nucleic acid.
The ferricyanide and ruthenium hexamine reporter system amplifies the electrochemical signal
from the redox reaction (Table 2). Ruthenium hexamine is a cationic electron acceptor, and
ferricyanide is an anionic electron acceptor23
. Ruthenium hexamine binds to the phosphate
backbone of nucleic acids via electrostatic interactions, and is reduced by the electrode6. The
reduction signal correlates to the amount of ruthenium hexamine present at the electrode, which
correlates to the amount of nucleic acid that hybridized6. Ferricyanide is electrostatically repelled
from the electrode due to its negative charge6. In solution, ferricyanide regenerates the oxidized
form of ruthenium hexamine, resulting in multiple redox cycles of ruthenium hexamine, which
amplifies the reduction signal (Table 2)6.
NME
Formation
Probe
Deposition
Background
Scan
Post hybridization
Scan
Hybridization
Ruthenium
Hexamine
Ferri
Cyanide
Peptide Nucleic
Acid (PNA)
DNA or RNA
Nanostructured
Microelectrode (NME)
Legend
A B C D E
9
Table 2: The components of the NME biosensing device.
Diagnostic techniques based on electrochemical measurements are faster, more
inexpensive, and require less data analysis algorithm than those based on optical and
mechanical measurements. The base pairing interactions of nucleic acid hybridization is
highly specific and relatively stable. This provides high assay selectivity compared to assays
that measure viral induced cell death. The ferricyanide and ruthenium hexamine reporter
system amplifies the electrochemical signal. Ferricyanide regenerates the oxidized form of
ruthenium hexamine, so the reduction of ruthenium hexamine can be measured multiple
times, thus amplifying the signal and increasing the assay sensitivity. The charge neutral
PNA probe increases the selectivity and sensitivity of the assay compared to a charged
DNA probe. Neutrally charged probe binds to nucleic acid stronger than charged probes,
due to the lack of charge repulsion. In addition, neutrally charged probe does not recruit
the electrochemical reporter, thus reducing the background signal. Nanostructured
microelectrode increases sensitivity relative to a bulk electrode, due to the larger surface
area available to attach probes.
The probe made from a PNA molecule decreases the background electrochemical signals (Table
2). PNA is a polymeric molecule composed of a peptide backbone with purine and pyrimidine
bases substituting for amino acid functional groups6. Moreover, PNA has higher affinity to the
complementary sequence than DNA or RNA due to the lack of charge repulsion6. In addition, the
neutrally charged peptide backbone cannot recruit ruthenium hexamine to the electrode without
the hybridization of the charged target sequence, resulting in the decrease of background signals
(Table 2)22
.
The NME increases the amount of probe sequences captured by the electrode and displayed to
the target sequence (Table 2). Nanostructures on the NME create more surface area and are more
efficient at capturing bio-molecules than bulk surfaces22
. This in turn increases the sensitivity of
the assay (Table 2)22
. The PNA probes are able to form a monolayer on the palladium surface of
10
the NME via palladium thiol bond22
. In addition, fabricating NMEs using photolithography and
electroplating generates reproducible structures rapidly and cost effectively22
.
Figure 3: The structure of a nanostructured microelectrode (NME).
A) The NME is approximately 20-30 µm in size. The NME structure is made of gold, since
gold is relatively durable. The surface of the NME is coated with palladium, since
palladium forms fine nanostructures. B) The nanostructures are shown as the grooves on
the branches of the electrode. The nanostructures increase the surface area, thus allowing
the attachment of more probes.
Figure 4: The top and cross-section of the NME biosensing chip.
A) The biosensing chips are manufactured in an array on a silicon wafer. Each chip
contains 20 gold leads. B) The bottom layer of the chip is the silicon wafer. The gold leads
are embedded in silicon dioxide above the silicon layer. An aperture is located at the tip of
each gold lead to expose the gold. The NME is seeded from the aperture, where the gold is
exposed. The other end of the gold lead is connected to the potentiostat, allowing the NME
to act as the working electrode.
Electrochemical based hybridization assays that utilize ferricyanide and ruthenium hexamine
reporter system, PNA, and NME provide the advantages of high sensitivity, high selectivity, high
speed, low detection limit, low cost, and low learning curve. The instrument that analyzes the
electrochemical signals is compact and inexpensive. Its low background signal and high signal
A B
Si
SiO2
Au Electrode
Aperture
A B
11
amplification allows it to have a 10aM detection limit 22
. This is achieved without molecular
labeling, PCR amplification, or multistep amplification procedures6. In addition, the NME
biosensing assay was shown to differentiate complementary from non-complementary sequences
in unpurified samples of lysed bacterial cells (Figure 5)24
. Moreover, the high sensitivity and
selectivity allow the discrimination of closely related non-complementary sequences in complex
biological samples25
.
Figure 5: The sensitivities and capabilities of the NME biosensor.
The NME biosensor is capable of differentiating between complementary and non-
complementary targets from unpurified samples24
In this case, bacterial lysates containing
nucleic acid, protein, lipid and cell debris are applied directly onto the chip without sample
purification.
Reprinted (adapted) with permission from (Lam, B., Fang, Z., Sargent, E. H. & Kelley, S.
O. Polymerase Chain Reaction-Free, Sample-to-Answer Bacterial Detection in 30 Minutes
with Integrated Cell Lysis. Analytical Chemistry 84, 21-25 (2011).). Copyright (2011)
American Chemical Society.
12
Chapter 2 Influenza Sequence Design and Assay Optimization
3 Overview
Detecting influenza with the NME biosensing assay required identifying suitable probe
sequences and optimizing the assay conditions. First, probes were designed to differentiate the
majority of influenza A strains from other sources of nucleic acids. Next, experiments were
conducted to control the quality of the probes and the quality of the biosensing chips. Finally,
different probe deposition conditions were tested to optimize the sensitivity and selectivity of the
assay.
4 Materials and Methods
4.1 Materials
Probe Synthesis. PNA probes were synthesized on a Prelude Peptide Synthesizer (Peptide
Instruments). The monomers for the synthesis were Fmoc-PNA-A(Bhoc)-OH (Link
Technologies 5001-C001), Fmoc-PNA-C(Bhoc)-OH (Link Technologies 5002-C001), Fmoc-
PNA-G(Bhoc)-OH (Link Technologies 5003-C001), Fmoc-PNA-T-OH (Link Technologies
5004-C001), Fmoc-L-Gly-OH (Peptide Instruments B-25-G), Fmoc-L-Cys(Trt)-OH (Peptide
Instruments B-25-CT), and Fmoc-L-Asp(OtBu)-OH (Peptide Instruments B-25-DB). Knorr
Resin (Advanced Chemtech SA5060), HATU (Advanced Chem Tech RC8110), 20% piperidine
in DMF (Peptide Instruments PS3-PPR-L), and 0.4NMM in DMF (Peptide Instruments PS3-
MM-L) were used as the resin, activator, deprotection agent, and base respectively. N,N-
dimethylformamide (Sigma-Aldrich D4551), dichloromethane (Sigma-Aldrich 443484), and
methanol (Sigma-Aldrich 179337) were the solvents used during the synthesis. Trifluoroacetic
acid (Sigma-Aldrich T6508), m-cresol (Sigma-Aldrich 65996), triisopropyl silane (Sigma-
Aldrich 233781), diethyl ether (Sigma-Aldrich 309966), and Bio-Spin Chromatography (Bio-
Rad) were used to process the probes after the synthesis.
Probe Purification. PNA probes were purified on an Agilent 1100 HPLC (Agilent
Technologies) with a Microsorb MV 300-5 C18 250*4.6mm HPLC column (Varian).
Dithiothreitol (Sigma-Aldrich 43815) and Spin-X centrifuge tube filters 0.22µm (Costar) were
13
used to treat the probes before the purification. Trifluoroacetic acid (Sigma-Aldrich T6508) and
acetonitrile (Sigma-Aldrich 360457) were the solvents used for the HPLC. Freezone 1
lyophilizer (Labconco) was used to lyophilize the probes.
Gel Binding Assay. Agarose (Bioshop AGA001.500), tris-borate-EDTA buffer 10X (Sigma-
Aldrich T4415-1L), and SYBR Gold nucleic acid gel stain (Invitrogen S-11494) were used to
make and to stain the gels. Owl Easycast B1AS Gel Runner (Thermo Scientific) and Power Pac
3000 power supply (Bio-Rad) were used to run the gels. Epichemi II Darkroom (UVP) was used
to image the gels.
Electrochemistry and Nucleic Acid Quantitation. Epsilon Potentiostat (BASi), silver/ silver
chloride reference electrode (BASi MF-2078), and platinum wire (Sigma-Aldrich 444685) were
used for all the electrochemistry experiments. All potentials were measured relative to the
reference electrode. The Nanodrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific)
was used to quantitate the nucleic acid solutions.
Chip Cleaning and Quality Control. The biosensing chip was custom made by AMS. AZ 300T
positive photoresist stripper (Clariant 300T), acetone (Sigma-Aldrich 650501), and isopropanol
(Sigma-Aldrich 19516) were used to chemically clean the surface of the chips. Reactive ion
etcher (SAMCO RIE-1C) and RF generator (ENI) were used to plasma etch the chips. Sulfuric
acid (Bioshop SUL001) and Eclipse LV100 Polarizing Microscope (Nikon) were used to check
for defects in the chips.
NME Formation. Gold (III) chloride solution (Sigma-Aldrich 484385), palladium (II) chloride
(Sigma-Aldrich 323373), hydrochloric acid (Bioshop HCL444), and perchloric acid (Sigma-
Aldrich 244252) were used to make the plating solutions for NME formation.
NME Biosensing Assay. UltraPure DNase/ RNase free distilled water (Invitrogen 10977015),
sonicator (Fisher Scientific FS60), digital heat block (Benchmark), and mercaptohexanol
(Sigma-Aldrich 725226) were used to prepare the probe solutions. Phosphate buffered saline
pH7.4 (Invitrogen 10010023), hexaammineruthenium(III) chloride (Sigma-Aldrich 262005), and
potassium hexacyanoferrate(III) (Sigma-Aldrich 244023) were used to prepare the scanning
solutions. Precision incubator (Thermo Scientific) was used for target hybridization.
14
4.2 Probe Sequence Design
The probes were designed using Influenza Primer Design Resource (www.ipdr.mcw.edu),
IDTDNA Oligo Analyzer (www.idtdna.com/analyzer/Applications/OligoAnalyzer/), and NCBI
BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi). Influenza Primer Design Resource was used to align
all the human influenza A sequences to identify conserved regions among different strains.
Regions that were at least 22-nucleotides in length and conserved in at least 90% of the strains
were selected as the raw sequence for probe design. The reverse complement of each region was
then generated. Next, each conserved region and its reverse complement were divided into 22-
nucleotide fragments as potential probe sequences. For example, a 24-nucleotide region would
yield 3 22-nucleotide fragments, and its reverse complement would also yield 3 fragments. The
22-nucleotide fragment length was chosen empirically to balance the high specificity of long
sequence lengths, and the synthesis efficiency of short sequence lengths.
Figure 6: The procedure for designing the probe.
First, influenza A sequences are aligned to locate a conserved region in the genome. The
probe sequence is selected from a conserved region to allow the probe to detect the
majority of influenza A strains. Next, the conserved region is divided into multiple 22-
nucleotide fragments, since 22-nucleotide length is empirically determined to be the ideal
probe length. This is because the length of 22 nucleotides balances probe specificity and
synthesis efficiency. Then, the segments are analyzed to find the sequences with low (more
negative) energy of hybridization, and high (less negative) energy of dimerization and
hairpin formation. Relative to alternative sequences, these are the sequences that have
higher affinity to the complementary molecule. Finally, the segments are analyzed to find
their similarity to human sequences. Eliminating sequences that are similar to human
sequences reduces false positives, since human sequences are the main source of nucleic
acid contamination for the assay.
IDTDNA Oligo Analyzer was used to determine the ΔG energy required for complementary
sequence hybridization, hairpin formation, and self-dimerization of each 22-nucleotide fragment.
The top sequences with the lowest (more negative) ΔGs of hybridization and the highest (less
Align influenza
sequences and select for
the most conserved
region
Divide region into 22nt segments
Check each segment for
the energy of hybridization, dimerization, and hairpin formation
Check the selected
segments for cross
hybridization with human sequences
15
negative) ΔGs of hairpin formation and self-dimerization were selected as potential probes for
further analysis.
NCBI BLAST was used to align the top potential probe sequences to human sequences from the
Human Genomic plus Transcript (Human G+T) database. The search was optimized for highly
similar sequences (Megablast). The potential probe sequences with the lowest sequence
similarity to human sequences were selected to be synthesized as PNA probes. The amino acid
residues Cys, Gly, and Asp were added to the PNA sequences prior to synthesis. Cys allowed the
attachment of the probe to the NME via thiol bond, Gly spaced the probe away from the NME
surface to prevent crowding during hybridization, and Asp increased probe solubility.
4.3 Probe Synthesis and Quality Control: PNA Synthesis
To set up the probe synthesis on the peptide synthesizer, a system wash with methanol was
performed. Next, N,N-Dimethylformamide (DMF) was used to dissolve the activator 333mM
HATU, and the monomers (100mM Fmoc-PNA-A(Bhoc)-OH, 100mM Fmoc-PNA-C(Bhoc)-
OH, 100mM Fmoc-PNA-G(Bhoc)-OH, 100mM Fmoc-PNA-T-OH, 100mM Fmoc-L-Gly-OH,
100mM Fmoc-L-Cys(Trt)-OH, and 100mMFmoc-L-Asp(OtBu)-OH). After that, the dissolved
activator and monomers, the wash solvent dichloromethane (DCM), the reaction solvent DMF,
the deprotection agent 20% piperidine in DMF, and the base 0.4M N-Methylmorpholine (NMM)
in DMF were poured into their respective solvent vessels. Finally, 0.35mg of the Knorr resin was
placed into the reaction vessel for each probe. During the synthesis, the first monomer was
attached to the resin using the swell coupling program, which had a long incubation period to
saturate the resin with the solvent, so that it could bind to the monomers. The subsequent
monomers were attached to the probe with the single coupling program with a short incubation
period, since the resin was already saturated with the solvent.
After the synthesis, the probes were processed on a vacuum manifold. First, the resins with
attached probes were washed and resuspended with DMF, and then transferred to a
chromatography column. Next, the column with the probes was washed with DMF, resuspended
with DCM, and washed with DMF again, to remove the contaminants. After that, the cleavage
solution containing 85% trifluoroacetic acid (TFA), 10% m-cresol, 2.5% distilled water, and
2.5% triisopropyl silane (TIPS) was incubated with the probes on a tilting platform for 2h, to
release the probes from the resin, and deprotect the PNA bases and amino acid side chains. Then,
16
the supernatant containing the probes was collected. Next, cold -80ºC diethyl ether was added to
the probe solution to precipitate the probes. After that, the mixture was centrifuged at 4000rpm at
-9ºC for 10min to pellet the probes, and the supernatant was then decanted. Then, the pellet was
resuspended with cold diethyl ether, and centrifuged 3 times to clean the probes. Finally, the
probes were allowed to dry overnight.
Figure 7: The procedure for synthesizing and characterizing the probe.
First, the PNA-peptide probe is synthesized by linking the monomers and processing the
resulting molecule. Then, the probe is purified on the HPLC to isolate the probe, and to
remove contaminants and incomplete probe fragments. Next, the purified probe is
analyzed on mass spectrometer to ensure that the resulting biological polymer is the
correct molecule. Finally, the probe is analyzed with the gel binding assay to ensure that
the resulting probe is capable of hybridizing to the complementary sequence.
4.4 Probe Synthesis and Quality Control: HPLC Purification and MS Analysis
To prepare the probes for HPLC purification, the cysteine residues on the probes were reduced
by incubating <1mg of the probe in a 0.5M dithiothreitol (DTT) 10% acetonitrile in water
solution for 1h. The solution was then centrifugally filtered at 13000rpm for 5min. Lastly, 100µl
of the sample was loaded onto the HPLC.
The HPLC mobile phase was made from the two solvents 0.1% TFA in acetonitrile, and 0.1%
TFA in distilled water. Initially, the mobile phase contained 5% of the acetonitrile solvent and
95% of the aqueous solvent. The acetonitrile concentration in the mobile phase increased over
time. The probes were initially purified using a steep acetonitrile gradient, where the acetonitrile
concentration increased 1.5% every minute. Due to poor separation of the probe from the side
products, a shallower gradient was used. In this gradient, the acetonitrile concentration increased
1.5% for 4min, then 0.25% for 20min, then 0.5% for 32min, and finally 1.5% for 4min. The
probes were then collected from the HPLC, lyophilized, and sent to SickKids Advanced Protein
Technology Centre for MS analysis.
Synthesize the PNA-peptide
probe
Purify the probe on HPLC
Confirm the probe mass
with MS
Check binding specificity with
gel binding assay
17
4.5 Probe Synthesis and Quality Control: Gel Binding Assay
The 5 different samples used to assay the quality of the probes were PNA probe alone,
complementary DNA alone, non-complementary DNA alone, PNA probe with complementary
DNA, and PNA probe with non-complementary DNA. All the samples were resolved on a 2%
agarose TBE gel in TBE buffer. DNA-alone samples were composed of 1X loading dye and
1µM DNA in PBS. PNA-alone samples were composed of 1X loading dye and 1µM PNA in
PBS. DNA-PNA hybridization samples were composed of 1X loading dye, 1µM PNA, and 1µM
DNA in PBS. All the samples were incubated at 37ºC prior to loading onto the gels. Next, the
gels were ran at 100V for 40min. After that, the gels were stained in a solution containing 1X
SYBR Gold in TBE for 30min. Finally, the gels were imaged.
4.6 Chip Cleaning and Quality Control: Chip Cleaning
The biosensing chips were manufactured on a silicon wafer. A layer of silicon dioxide was
deposited on the silicon wafer, and gold leads were deposited on top of the silicon dioxide layer.
The chips were then passivated with a layer of silicon dioxide to insulate the leads. Next, 500nm
diameter apertures were created at the tip of each lead to allow for NME formation. Finally, each
chip was protected with a positive photoresist.
To clean a chip, it was first incubated in AZ 300T photoresist stripper for 5min. The chip was
then rinsed with acetone, then isopropanol, and finally water. After that, the chip was dried with
a stream of nitrogen gas. Next, the chip was etched with an oxygen plasma etcher at 15W for
120s. Finally, the chip was rinsed with acetone, isopropanol, and water.
Figure 8: The procedure for characterizing and cleaning the chip.
First, the chip is treated with the AZ 300T stripper to remove the photoresist. This
treatment exposes the gold lead under the aperture. Then, the chip is washed with acetone,
isopropanol, and distilled water to remove the stripper and dust particles. Next, the chip is
etched with plasma to remove organic particles, and to make the surface hydrophilic. After
that, the chip is washed with acetone, isopropanol, and distilled water again to remove dust
and contaminants. Finally, the leads are examined with acid scan to find defect free and
contaminant free electrodes with proper sized aperture.
AZ 300T treatment
Acetone-isopropanol
-distilled water wash
Plasma etch
Acetone-isopropanol
-distilled water wash
Acid scan
18
4.7 Chip Cleaning and Quality Control: Acid Scan
Each chip was connected to the Epsilon Potentiostat and acted as the working electrode. A silver/
silver chloride electrode acted as the reference electrode, and a platinum wire acted as the
counter electrode. The potentiostat was used to scan the chips with either Cyclic Voltammetry
(CV) or Differential Pulse Voltammetry (DPV). CV was ideal for examining the quality of the
NME and the quality of the deposited probe. DPV was ideal for quantitating the electrochemical
signal.
To assay the quality of the leads on the chip, the chip and the electrodes were immersed in a
50mM sulfuric acid solution. The chip was then scanned with CV. CV was set to scan from an
initial potential of -0.1V to a switching potential of 1.5V, at a scan rate of 100mV/s.
4.8 NME Formation: Microelectrode Formation
The gold NMEs were electroplated by submerging a cleaned chip, the reference electrode, and
the counter electrode in a gold plating solution containing 50mM gold chloride and 500mM
hydrochloric acid. Direct Current Potential Amperometry (DCPA) was used to electroplate the
gold leads by applying a relative potential (potential relative to the reference electrode) of 0V for
30s. A clean defect free chip would produce a ~20µA final current and a ~30µm NME. The size
of the NME was checked under a light microscope at 10X magnification.
4.9 NME Formation: Nano Structure Formation
The nanostructures on the NME were formed by submerging a chip with gold NME in a
palladium plating solution containing 5mM palladium chloride and 500mM perchloric acid.
DCPA was used to form the nanostructures by applying a relative potential (potential relative to
the reference electrode) of -0.25V for 5s. A defect free NME would produce a ~4µA final
current.
4.10 NME Biosensing Assay: Probe Deposition
The PNA probes were co-deposited with mercaptohexanol (MCH). The deposition solution was
composed of 0.1µM probe and 0.9µM MCH dissolved in a solvent of 10% acetonitrile in
distilled water. The solution was sonicated for 30min, and heated to 60ºC for 30min to dissolve
the probe. Next, a 30µl volume of the deposition solution was deposited onto a chip, and
19
incubated in a humidity chamber for 1h at room temperature. The chip was then washed with
PBS 3 times, and incubated in 10% PBS until it was ready for the electrochemical assay.
4.11 NME Biosensing Assay: Pre Hybridization Scan
The sensing solution was composed of 4000µM potassium hexacyanoferrate(III) and 10µM
hexaammineruthenium(III) chloride in 10% PBS. Before adding the hexacyanoferrate(III) and
the hexaammineruthenium(III), the 10% PBS was purged by bubbling nitrogen gas through the
10% PBS for 30min. In addition, the sensing solution was prepared fresh every 30min. A chip
with probe modified NME was incubated in the sensing solution for 1min, and then scanned with
DPV. The chip was scanned from 0V to -0.4V with 5mV step E, 50ms pulse width, 100ms pulse
period, and 10mV pulse amplitude. The pre hybridization scan current (I1) was measured from
the baseline to the peak of the current (Figure 9). The chip was then incubated in PBS until it was
ready for hybridization.
4.12 NME Biosensing Assay: Hybridization
The target solution was prepared by diluting nucleic acid (DNA or RNA) in PBS. A 30µl volume
of the target solution was applied to the probe modified NME, and incubated at 37ºC for 30min.
The chip was then washed 3 times with PBS, and incubated in 10% PBS until it was ready for
scanning.
4.13 NME Biosensing Assay: Post Hybridization Scan
The post hybridization scan used the same scanning solution preparation method, the same DPV
parameters, and the same current measurement as the pre hybridization scan. The post
hybridization current was defined as I2 (Figure 9). The baselines of the pre and the post
hybridization DPV scans were adjusted using Matlab by aligning them to the horizontal plane. In
this way, the DPV scans were superimposed (Figure 9). The change in current (ΔI) was
calculated by subtracting the pre hybridization current from the post hybridization (ΔI= I2- I1)
(Figure 9). Due to changes in the reduction potential, the binding of the complementary sequence
could shift the electrochemical signal to a higher or lower voltage relative to the signal produced
from the probe alone. However, the electrochemical signal always resided close to the -200mV
region.
20
The change in signal intensity of the DPV scans was mainly caused by the reduction of the
reporter molecules, that were recruited via the binding of the target sequence. However, the
signal intensity could also be affected by the quality of the NME, the size of the NME, the
quantity of the probe deposited onto the NME, the quality of the probe, the probe to MCH ratio,
the incubation time for hybridization, the chemistry of the scanning solution, the quality of the
electrodes, the contact between the chip and the potentiostat, and the potentiostat. Negative
control experiments using non-complementary target sequences were performed to account for
the signal change caused by sources other than target sequence hybridization. Negative controls
were conducted in parallel with the test samples using the same potentiostat, electrodes, scanning
solution, chip, probe, deposition solution, deposition conditions, and incubation conditions.
Figure 9: A Differential Pulse Voltammetry (DPV) plot used to assay for target
hybridization.
The probe covered electrode is scanned with DPV to determine the background current.
The background current is created by the reduction of the electrochemical reporters
associated to the electrode and probe. This blue curve is the pre target hybridization signal,
and the associated current is measured as I1. The probe covered electrode that hybridized
with the target nucleic acid is scanned with DPV to determine the signal current. The signal
current is generated by the reduction of the electrochemical reporters recruited by the
target sequence. This red curve is the post hybridization signal, and the associated current
is measured as I2. The change in current (ΔI) is calculated by subtracting pre hybridization
peak height from the post hybridization peak height. The ΔI represents the change in
current induced by the hybridization of the target sequence.
Δ I
=(I2-I1)
I1
I2
Potential (V)
Cu
rren
t (A
)
21
4.14 Probe Deposition Condition Optimization
The methods from sections 4.6 to 4.13 described the assay procedure using the optimized probe
deposition condition. However, 3 additional deposition conditions were also examined. The 4
deposition solutions were 0.1µM probe and 0.9µM MCH in 10% acetonitrile solution (1µM total
concentration, 1:9 probe to MCH ratio), 0.5µM probe and 0.5µM MCH in 10% acetonitrile
solution (1µM total concentration, 1:1 probe to MCH ratio), 0.5µM probe and 4.5µM MCH in
10% acetonitrile solution (5µM total concentration, 1:9 probe to MCH ratio), and 2.5µM probe
and 2.5µM MCH in 10% acetonitrile solution (5µM total concentration, 1:1 probe to MCH
ratio). The probe 2A was used to optimize the deposition conditions. For the hybridization step,
the chips were incubated with either 100nM complementary DNA (positive control) or 100nM
non-complementary DNA (negative control).
5 Results and Discussion
5.1 Probe Sequence Selection
The probes for the NME biosensing assay were designed to differentiate sequences from the
majority of influenza A strains apart from nucleic acids originating from other sources. This
process involved identifying a conserved region to design potential probe sequences, checking
the thermodynamic properties of the potential probes, and checking for cross hybridization of the
potential probes with human sequences.
During its life cycle, influenza produces the coding strand RNA in the form of mRNA
transcripts, and the non-coding strand RNA in the form of genomic segments8. Therefore, probes
complementary to the influenza genome and probes complementary to the influenza transcripts
were both designed, in order to maximize the amount of influenza RNA that could be detected.
The probes should target a conserved influenza sequence, so that they could detect influenza A
RNA even though the most prevalent influenza strain differed each year. More than 9000
influenza A sequences were aligned to locate the most conserved region: 5’-TTT GTG TTC
ACG CTC ACC GTG CCC AGT GAG CGA GGA CTG CAG CGT AGA CGC TTT GTC CAA
AAT GCC CT-3’. This region had 68 nucleotides and was found on the matrix gene in segment
7 of the influenza genome. This conserved sequence was located on the coding strand. Thus, it
22
was used to make the genome binding probe. The reverse complement of this conserved region
was located on the non-coding strand. Thus, it was used to make the transcript binding probe.
PNA is more hydrophobic than both DNA and RNA, since it does not have the charged
phosphate backbone22
. The ideal probe length was found empirically to be ~22 nucleotides, since
longer probes were difficult to synthesize and might aggregate, while shorter probes might not be
specific. The 68-nucleotide conserved region and its reverse complement were both divided into
47 22-nucleotide segments as potential probes.
Thermodynamically, the ideal probe has high affinity to its complementary sequence and low
affinity to non-complementary sequences, so that it is specific and sensitive. It does not form
hairpins nor dimerize, since probe with hairpins and probe dimers do not bind target nucleic acid
effectively. Therefore, probes with low (more negative) ΔGs of hybridization and high (less
negative) ΔGs of hairpin formation and dimerization were selected as potential probes for further
analysis. IDT DNA Oligo Analyzer tool was used to calculate the ΔGs of hybridization,
dimerization, and hairpin formation to find the most energetically optimal sequences.
In a bronchial alveolar lavage sample, the main source of nucleic acid is from human cells26
.
Therefore, potential probes with good thermodynamic properties were aligned to human
sequences to check for cross hybridization. The potential probes that had the least similarities
with human sequences were selected for further modification. The nucleotide sequence of the
final genome binding probe was 5’-CGT GCC CAG TGA GCG AGG ACT G-3’ (Table 3). The
nucleotide sequence of the final transcript binding probe was 5’-AGG GCA TTT TGG ACA
AAG CGT C-3’ (Table 3). The sequences of the selected probes originated from the same region
of the influenza genome as the influenza A H1N1 universal probe sequence designed by CDC27
.
In addition to the 22 PNA residues, 4 amino acids were added to both probes. The sequence Cys-
Gly-Asp was added to the 5’ end of the probe, and an Asp residue was added to the 3’ end of the
probe. The Cys residue had a thiol group, which was important for attaching the probe to the
palladium coated NME. The Gly residue was used to increase the distance between the PNA
bases and the NME, so that the probe sequence was physically more exposed to the target
nucleic acids present in the diagnostic samples. The 2 Asp residues added charges to the probe,
so that the probe was more soluble in PBS.
23
Table 3: The properties of the selected probe sequences.
Compare to all potential probes, the selected probes have low (more negative) ΔG of
hybridization to maximize targeting binding, and high (less negative) ΔG of hairpin and
dimer formation to minimize self-hybridization. In addition, they have low sequence
similarity with all human sequences to minimize cross-hybridization.
5.2 Probe Quality Control
The quality of the probes was checked to ensure that the probes could hybridize to their target
sequences. HPLC was used to separate the probes from side products. MS was used to confirm
the mass of the probes. Gel binding assay was used to analyze the binding specificity of the
probes.
The absorbance chromatogram at 260nm from the HPLC analysis showed several peaks,
suggesting that the probe synthesis process generated side products along with the probe (Figure
11). Purification of PNA from the HPLC column depended on the acetonitrile concentration,
thus modifying the rate of change of the acetonitrile concentration altered the time that molecules
exited from the column. Initially, a steep acetonitrile gradient (acetonitrile concentration
increased at a rate of 1.5% per minute), was used to purify the probe (Figure 10). This resulted in
narrow peaks and a short purification time, where the major peak appeared around 15min (Figure
11). However, several of the peaks overlapped, suggesting that the probe and the side products
were still mixed after the purification (Figure 11). A modified method with a shallower
acetonitrile gradient was used to increase the resolution of the HPLC spectrum (Figure 10). In
24
this method, acetonitrile concentration increased 1.5% every minute for the first 4min, because
nothing exited the column in this range of acetonitrile concentrations. Acetonitrile concentration
increased 0.25% every minute for the next 20min to maximize the separation of the different
products. In the following 32min, acetonitrile concentration increased 0.5% every minute to
sharpen the peaks, since the probe was purified from the column during this range of acetonitrile
concentrations. The acetonitrile concentration increased 1.5% every minute for the final 4min to
flush out any products left in the column. The HPLC method with the shallow gradient
significantly improved the separation of the probe from the side products (Figure 11).
Figure 10: The acetonitrile gradient of the HPLC methods used to purify the probes.
The purification of the probe via HPLC depends on the acetonitrile concentration of the
mobile phase. The 22-nucleotide probe exits the column at ~20% acetonitrile concentration.
In addition to the probe, the probe synthesis process also generates incomplete fragments
and side products. When purifying with a steep acetonitrile gradient, the probe exits the
column very quickly, but side products also exit the column with the probe. An improved
gradient is designed so that it is very steep at the beginning of the purification, since
nothing exists at the beginning. The gradient between 10% and 20% acetonitrile
concentration is very shallow to separate the side products from the probe. Finally, the
gradient near 20% acetonitrile concentration is slightly steeper, so that the probe is easy to
visualize on the chromatogram.
The products exiting from the HPLC column were collected every 2min and analyzed with MS.
For the 1C probe, the peak corresponding to the mass of the probe was found in the 40-42min
and the 42-44min fractions (Figure 11). For the 2A probe, the peak corresponding to the mass of
the probe was found in the 36-38min and the 38-40min fractions (Figure 11). For both probes,
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70
Ace
ton
itri
le c
on
cen
trat
ion
in m
ob
ile
ph
ase
(%
)
Time (min)
Steep acetonitrile gradient Shallow acetonitrile gradient
25
these fractions corresponded to the location of the largest peak on their HPLC chromatograms.
For both probe purifications, these results thus showed that the major product was indeed the
probe.
Figure 11: The HPLC chromatograms showing the purification of the probes.
The purification of the probe is visualized by tracking the absorbance at 260nm, since the
bases on PNA absorb at this range. The probe exits the column at ~20% acetonitrile
concentration, thus the probe is recovered around 15 min in the steep gradient and around
40 min in the shallow gradient. Diagrams A and B show the purification of probe 1C on the
steep and the shallow gradient, respectively. A) Probe 1C purified in the steep acetonitrile
gradient produces a ~2500mAU peak, since side products are superimposed with the probe.
B) Probe 1C purified in the shallow acetonitrile gradient produces a ~600mAU peak, since
the peaks are more resolved and most of the side products exit the column before probe 1C.
Diagrams C and D show the purification of probe 2A on the steep and the shallow gradient,
respectively. C) Probe 2A purified in the steep acetonitrile gradient produces a ~1500mAU
peak, since side products are superimposed with the probe. D) Probe 2A purified in the
shallow acetonitrile gradient produces a ~600mAU peak, since the peaks are more resolved
and most of the side products exit the column before probe 2A.
0
500
1000
1500
2000
2500
0 5 10 15 20Ab
sorb
ance
at
26
0n
m
(mA
U)
Time (min)
0
200
400
600
0 20 40 60Ab
sorb
ance
at
26
0n
m
(mA
U)
Time (min)
0
500
1000
1500
2000
2500
0 5 10 15 20Ab
sorb
ance
at
26
0n
m
(mA
U)
Time (min)
0
200
400
600
0 20 40 60Ab
sorb
ance
at
26
0n
m
(mA
U)
Time (min)
A B
C D
26
Figure 12: The gel binding assay confirming the specific hybridization of the PNA probes
to the target DNA.
The gel binding assay uses the SYBR Gold dye, which binds to single or double stranded
DNA, but not peptide nucleic acid (PNA) nor DNA-PNA hybrids. Relative to the DNA
target alone, DNA-PNA hybrids stain lighter and migrate slower. However, DNA and PNA
fragments that are incubated together but do not bind are located at similar position and
have similar band intensity as the DNA fragment alone. This assay is performed on the
HPLC fractions (1C40, 1C42, 2A36, and 2A38) that contain the probe. As expected, the
PNA probe alone samples (negative control) have no band and the DNA alone samples
(negative control) have bright fast migrating bands. All non-complementary DNA with
probe samples have fast migrating bands, demonstrating that the probes do not bind to the
non-complementary sequences. All complementary DNA with PNA probe samples have
slow migrating faint bands, indicating that the probes bind to the complementary
sequences.
27
The gel binding assay tested for the ability of a PNA sequence to hybridize to the complementary
DNA sequence (Figure 12). The hybridization temperature and incubation period used for the gel
binding assay were the same as the corresponding conditions used for the NME biosensing
assay, so that the results from the gel binding assay were relevant to the NME biosensing assay.
The SYBR Gold dye binds double or single stranded DNA but not PNA or DNA-PNA hybrids.
Moreover, when DNA and PNA hybridize, they migrate slower than DNA alone. Therefore,
when the PNA probe hybridizes to a complementary DNA sequence, the band is fainter and
migrates slower than the DNA alone band. However, when the PNA probe is incubated with a
non-complementary DNA sequence, the band is the same intensity and migrates to the same
location as the DNA alone band. The gel binding assay was performed on the HPLC fractions
(1C40, 1C42, 2A36, and 2A38) that contained the probe (Figure 12). As expected, all probe
alone samples had no band, and all DNA alone samples had bright fast migrating bands (Figure
12). All non-complementary DNA with probe samples had fast migrating band, indicating that
the probe did not hybridize to non-complementary DNA (Figure 12). All complementary DNA
with probe samples had slow migrating faint bands, since the hybridization of the probe to the
complementary DNA inhibited the binding of the dye (Figure 12).
5.3 Chip Quality Control
The AZ 300T treatment was used to remove the positive photoresist. The acetone, isopropanol,
and water rinses were used to clean the chip. The plasma etch was used to increase the
hydrophilicity of the chip surfaces. The removal of the photoresist exposed the gold surfaces in
the aperture, so that the NME could be electroplated. The generation of a hydrophilic surface
prevented non-specific deposition of the hydrophobic PNA probe onto the chip surface. Acid
scan was used to ensure that the leads were defect free, and that the gold surfaces were free from
contaminants. During the acid scan in 50mM sulfuric acid, the oxidation and reduction of a clean
gold surface created peaks at 1.2V and 0.9V, respectively (Figure 13A). The absence of these
peaks, the presence of other peaks, or any distortion in the Cyclic Voltammetry graph indicated
an unclean surface or a defective lead (Figure 13B).
28
Figure 13: The acid scan checking the gold leads for defects and contaminants.
Gold oxidizes at 1.2V and reduces at 0.9V in 50mM sulfuric acid, resulting in peaks at these
potentials. Absence of these features indicates the presence of contaminants or defects in
the lead. A) Diagram A shows the acid scan of a clean and defect free lead. The scan of a
clean lead has relatively small current change throughout the scan except at the 1.2V and
the 0.9V potentials. B) Diagram B shows the acid scan of a defective or unclean lead. The
large current change suggests that the lead possibly has multiple apertures or
contaminants. Leads like this are not used.
5.4 Probe Deposition Condition Optimization
The ideal deposition condition should result in high signal to background ratio at low target
molecule concentrations. The deposition solution was composed of the probe and MCH, where
the MCH binds to the NME competitively and allows the probe to form an evenly spaced
monolayer. Thus, deposition required the optimization of the probe and the MCH concentrations.
High probe concentrations could result in high background signal, while low probe
concentrations could provide fewer sites for target nucleic acid to bind. Moreover, high MCH
concentrations could reduce the number of probes bound to NME, while low MCH
concentrations could cause the probe to clump on the NME and increase the background signal.
-4.00
-2.00
0.00
2.00
4.00
-100 300 700 1,100 1,500
Cu
rre
nt
(nA
)
Potential (mV)
-100.00
-50.00
0.00
50.00
100.00
-100 300 700 1,100 1,500
Cu
rre
nt
(nA
)
Potential (mV)
A B
29
Figure 14: The NME biosensing assay comparing different deposition conditions.
Probe 2A is used for this experiment. The black bars represent hybridization to a
complementary DNA target, while the white bars represent hybridization to a non-
complementary DNA target. Mercaptohexanol (MCH) is a reagent that prevents probe
aggregation on the electrode surface. The probe:MCH ratio and the total probe-MCH
concentration are varied in the experiment. Comparing the deposition conditions with the
same total probe-MCH concentration and different probe:MCH ratios, the condition with
the higher MCH concentration (1st and 3
rd set of bars) results in a higher signal to noise
ratio. When comparing the deposition conditions with the same probe:MCH ratio and
different total probe-MCH concentrations, the condition with the lower total concentration
(1st and 2
nd set of bars) results in a higher signal to noise ratio. Thus, in the conditions
tested, 1µM total probe MCH concentration with a 1:9 probe:MCH ratio is the optimal
condition that maximizes the hybridization of complementary sequences and minimizes the
non-specific binding of non-complementary sequences.
Deposition solution composition was optimized by varying both the combined probe and MCH
concentration, and the probe to MCH ratio. The 4 conditions tested were 1µM total concentration
in 1:9 probe:MCH ratio, 1µM total concentration in 1:1 probe:MCH ratio, 5µM total
concentration in 1:9 probe:MCH ratio, and 5µM total concentration in 1:1 probe:MCH ratio
(Figure 14). Comparing the deposition conditions with the same total probe-MCH concentration
and different probe:MCH ratios, the condition with the higher MCH concentration resulted in a
higher ΔI when incubated with complementary DNA, and a lower ΔI when incubated with non-
complementary DNA (Figure 14). When comparing the deposition conditions with the same
probe:MCH ratio and different total probe-MCH concentrations, the condition with the lower
total concentration resulted in a higher ΔI when incubated with complementary DNA, and a
lower ΔI when incubated with non-complementary DNA (Figure 14). Thus, in the conditions
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
1µM 1:9Probe:MCH
ratio
1µM 1:1Probe:MCH
ratio
5µM 1:9Probe:MCH
ratio
5µM 1:1Probe:MCH
ratio
ΔI (
nA
)
Deposition Solution Composition
30
tested, 1µM total probe MCH concentration with a 1:9 probe:MCH ratio was the optimal
condition that maximized the hybridization of complementary sequences and minimized the non-
specific binding of non-complementary sequences (Figure 14).
31
Chapter 3 Influenza Biosensing
6 Overview
The NME biosensing assay was tested to detect the target influenza sequence in the forms of
synthetic DNA, in vitro transcribed RNA, and RNA released from cell lysate. Target DNAs were
assayed to check the ability of the NME biosensing assay to differentiate complementary from
non-complementary sequences. In vitro transcribed RNAs were assayed to determine if the NME
biosensing assay could detect large RNA molecules, since influenza nucleic acids were large
RNA molecules. Finally, transfected cell lysates that contained influenza RNA were assayed to
simulate patient bronchial alveolar lavage samples.
7 Materials and Methods
7.1 Materials
Target Sequence Samples. DNA oligonucleotide 1C_T 5’-CAG TCC TCG CTC ACT GGG
CAC G-3’(IDTDNA), DNA oligonucleotide 2A_T 5’-GAC GCT TTG TCC AAA ATG CCC T-
3’(IDTDNA), and DNA oligonucleotide EC_T 5’-AAC TAC ACC ACA GAG CAG AT-
3’(IDTDNA) were used as the DNA targets. FirstChoice human lung total RNA (Ambion
AM7968) was used as the RNA negative control target.
In vitro Transcription. BamHI (Thermo Scientific ER0051) was used for the restriction digest.
Phenol: chloroform: isoamyl alcohol 25:24:1 10mM tris pH8 1mM EDTA (Sigma-Aldrich
P2069), sodium acetate ph5.2 buffer solution (Sigma-Aldrich S7899), ethanol (UofT Medstore
39752-PO16-EAAN), and UltraPure DNase/ RNase free distilled water (Invitrogen 10977015)
were used to extract and precipitate DNA. T7 RiboMAX Express Large Scale RNA Production
System (Promega P1320), phenol: chloroform: isoamyl alcohol 125:24:1 (Sigma-Aldrich
77619), 2-propanol (Sigma-Aldrich I9516), and ethanol (UofT Medstore 39752-PO16-EAAN)
were used to in vitro transcribe and purify RNA.
Cell Transfection. A549 Human Carcinomic Human Alveolar Basal Epithelial cell line (ATCC
CCL-185) was the cell line used for transfection. Ham’s F-12K medium (Invitrogen 21127022),
fetal bovine serum (Invitrogen 12483020), and Forma Series II Water Jacketed CO2 Incubator
32
(Thermo Scientific) were used to culture the A549 cells. Phosphate buffered saline pH7.4
(Invitrogen 10010023), 0.25% trypsin EDTA phenol red (Invitrogen 25200056), X-tremeGENE
HP DNA transfection reagent (Roche 06366236001), and Opti-MEM reduced-serum medium
(Invitrogen 31985062) were used to transfect the A549 cells. TC10 automated cell counter (Bio-
Rad) was used to quantify the cells. OmniLyse lysis kit (ClaremontBio 01.340.24) was used to
lyse the cells. RNeasy Mini Kit (Qiagen 74104) and ethanol (UofT Medstore 39752-PO16-
EAAN) were used to purify the cellular RNA.
RNA Quantitation. RTPCR and QPCR were performed on a MyCycler Thermal Cycler (Bio-
Rad) and a 7500 Fast Real Time PCR System (Applied Biosystems) respectively. High Capacity
RNA-to-cDNA kit (Invitrogen 4387406), and Power SYBR Green PCR Master Mix (Applied
Biosystems 4367659) were the master mixes used for RTPCR and QPCR respectively. DNA
oligonucleotide primer_F 5’-GAC CAA TCT TGT CAC CTC TGA C-3’(IDTDNA) and DNA
oligonucleotide primer_R 5’-AGG GCA TTT TGG ACA AAG CGT CTA-3’(IDTDNA) were
the primers used for QPCR.
7.2 Target Vector Design
To generate the target RNA sequence, 2 plasmids were designed and synthesized. One expressed
the influenza segment 7 genomic RNA and the other expressed the influenza matrix transcript
RNA. The sequence of the Influenza A Virus (A/Hong Kong/H090-770-V22/2009(H1N1))
matrix protein 2 (M2) and matrix protein 1 (M1) gene (Genebank accession number
CY120390.1) was used as the starting sequence to design the plasmid that encoded the influenza
matrix transcript RNA. The reverse complement of this sequence was used as the starting
sequence to design the plasmid that encoded the influenza segment 7 genomic RNA. Both
sequences were modified by adding a BamHI restriction site at their 3’ ends and a HindIII
restriction site at their 5’ ends to allow for subcloning. The modified sequences were sent to
GeneArt to be synthesized and subcloned into pcDNA1.3(+) plasmids. GeneArt confirmed the
sequences of the plasmids with DNA sequencing and restriction digest.
7.3 In vitro Transcription
Restriction digest was completed in a reaction of volume of 40µl to prepare the plasmid for in
vitro transcription. The reagents were added in the following order: 30µl of nuclease free water,
33
4µl of 10X Buffer BamHI, 2µl of 1µg/µl plasmid DNA (restriction digest was performed for the
plasmids vec_inf_ma_genome_pcDNA3.1+ and vec_inf_ma_genemrna_pcDNA3.1+), and 4µl
of BamHI. The mixture was incubated at 37ºC for 2h.
The linearized plasmid DNA was extracted by adding an equal volume of phenol: chloroform:
isoamyl alcohol 25:24:1 10mM tris pH8 1mM EDTA solution to the restriction digest. Then, the
mixture was vortexed and centrifuged at 1300rpm at room temperature for 30s. After that, the
aqueous phase was mixed with one tenth volume of 3M sodium acetate. Next, two fold volume
of cold 95% ethanol was added. After that, the mixture was centrifuged at 1300rpm for 5min,
and the supernatant was removed. Then, 1ml of 70% ethanol was added to the pellet, and
centrifuged at 1300rpm for 5min. Finally, the supernatant was decanted, and the pellet was air
dried and resuspended in UltraPure DNase/ RNase free distilled water.
The purified linearized plasmid DNA was in vitro transcribed using T7 RiboMAX Express Large
Scale RNA Production System according to the manufacture’s protocol.
7.4 Cell Transfection
A549 cells were cultured, and transfected with the plasmids vec_inf_ma_genome_pcDNA3.1+
and vec_inf_ma_genemrna_pcDNA3.1+. A549 cells were cultured in 10% fetal bovine serum
(FBS) Ham’s F-12K medium (F-12K), and seeded in 6-well plates at a concentration of 105
cell/ml. The cells were incubated at 37ºC and 5% carbon dioxide until they were grown to 80%
confluence. The media was renewed 12h before transfection.
To transfect the cultured A549 cells, 5µl of 1µg/µl plasmid DNA (transfection was performed
for the plasmids vec_inf_ma_genome_pcDNA3.1+ and vec_inf_ma_genemrna_pcDNA3.1+)
was added to 500µl of Opti:MEM reduced-serum medium, and mixed. Next, 5µl of X-
tremeGENE HP DNA transfection reagent was added to the transfection mixture. The
transfection mixture was then incubated at room temperature for 30min, and added to the
cultured A549 cells without changing media. For the negative control, the transfection mixture
did not contain any plasmid DNA. The cells were then incubated at 37ºC and 5% carbon dioxide
for 24h.
34
7.5 Cell Lysis and Lysate Preparation
The cells were harvested after transfection by removing the old media, and replacing it with
0.25% trypsin EDTA phenol red. Then, the cells were incubated at room temperature for 10min.
Next, the cells were centrifuged at 5000rpm. After that, the supernatant was decanted, and the
cells were washed and resuspended in PBS. Then, the cell concentration was measured with an
automated cell counter, and then diluted to 5*104 cells/ml. After that, the suspended cells were
lysed with OmniLyse lysis kit. Next, the lysates were aliquoted, where some were reserved for
QPCR and some were reserved for the NME biosensing assay. The RNA in the aliquots reserved
for QPCR was purified with RNeasy Mini Kit according to the manufacture’s protocol. Once the
concentrations of the influenza genomic RNA and the influenza transcript RNA were quantitated
with QPCR, the lysates that contained the influenza RNA were diluted to 106 copies of influenza
RNA/µl using the lysates that did not contain any influenza RNA, so that the total cellular RNA
concentration remained constant.
7.6 QPCR Analysis
The absorbances of the in vitro transcribed RNA of both influenza genomic RNA and influenza
transcript RNA were measured with a Nanodrop UV-Vis spectrophotometer, and their
concentrations were calculated. Both RNA samples were serial diluted with PBS to 1010
, 109,
109, 10
7, 10
6, 10
5, and 10
4 copies/µl. These samples were then used to construct the standard
curve for the QPCR assay. The standard curve samples, the influenza transcript and genomic
RNAs from the lysed transfected cells, the RNA from lysed but not transfected cells, and 3
QPCR negative controls (no template, no primer, and no enzyme) were reverse transcribed using
the High Capacity RNA-to-cDNA kit, according to the manufacture’s protocol. The cDNA
products were then quantitated with the Power SYBR Green PCR Master Mix, using 200nM of
primers and following the manufacture’s protocol.
8 Results and Discussion
8.1 Target Vector Design
Plasmids that encoded the influenza RNA were designed to generate a safe non-biohazardous
source of influenza samples. Two different plasmids were constructed, where one encoded
influenza genomic segment 7 RNA and the other encoded influenza matrix transcript RNA
35
(Figure 15). The pcDNA3.1+ plasmid contained a T7 promoter and a BamHI restriction site, so
large quantities of RNA could be produced via in vitro transcription (Figure 15). The plasmid
also contained a CMV promoter and a BGH poly (A) site, so that it could be transcribed in a
human lung cell (Figure 15).
Figure 15: The plasmids for generating the influenza RNA sequences.
The pcDNA3.1+ plasmid is used to create both plasmid constructs. It contains a T7
promoter and a BamHI cleavage site, so that transcripts can be generated via in vitro
transcription. The CMV promoter and BGH poly (A) site allow the transcription of the
sequence in human lung cells. The target sequences are inserted via the HindIII and
BamHI sites. A) Diagram A shows the plasmid used to create the influenza genome
segment 7 via in vitro transcription. B) Diagram B shows the plasmid used to create the
influenza matrix gene transcript via in vitro transcription.
A
B
36
8.2 DNA Target Biosensing
The NME biosensing assay was performed on 22-nucleotide target DNA oligonucleotides. The
hybridization of the probes to the oligonucleotides was measured with DPV scans (Figure
16).This experiment was conducted to ensure that the assay was capable of differentiating
complementary from non-complementary sequences. Several different concentrations of the
target DNA were used to determine the detection limit of the assay. DNA rather than RNA was
assayed even though influenza genome and transcripts are RNA molecules, since DNA is more
stable1.
Figure 16: Representative DPV scans using the NME biosensing assay.
A) Diagram A shows a DPV scan from assaying a sequence complementary to the probe.
The current from the post hybridization scan is much greater than the current from the
pre hybridization scans, since the hybridized DNA recruits reporter molecules to the NME
to amplify the signal. B) Diagram B shows a DPV scan from assaying a sequence not
complementary to the probe. The current from the post hybridization scan is similar to the
current from the pre hybridization scan, since the non-complementary DNA does not bind
to the probe, and thus does not recruit reporter molecules to the NME.
Both probes 1C and 2A were examined with the biosensing assay. The negative control for both
probes was a non-complementary Escherichia coli sequence (Figure 17). The 10000pM, 100pM,
and 1pM concentrations of the complementary DNA target were assayed for both probes (Figure
17). The concentration of the negative control target was at least ten fold greater than the
concentration of any of the complementary targets (Figure 17). In this experiment, 5 independent
trials were performed for each test condition.
In the trials using probe 1C, the mean ΔI for the 100mM Neg Ctrl, the 10000pM, 100pM, and
1pM samples were 0.198nA, 3.58nA, 2.78nA, and 0.912nA, respectively. The standard error of
0.0
0.5
1.0
1.5
2.0
2.5
3.0
-400 -300 -200 -100 0
Cu
rre
nt
(nA
)
Potential (mV)
Pre hybridization Post hybridization
0.0
1.0
2.0
3.0
-400 -300 -200 -100 0
Cu
rre
nt
(nA
)
Potential (mV)
Pre hybridization Post hybridization
A B
37
the mean for the 100mM Neg Ctrl, the 10000pM, 100pM, and 1pM samples were 0.0632nA,
0.259nA, 0.307nA, and 0.127nA, respectively. The data were statistically significant, since the
standard error bar of the negative control did not overlap with the ones from the trials that used
complementary DNA sequence (Figure 17). The precision of the assay was expressed via relative
standard error of the mean. The relative standard error of the mean for the negative control was
32%. This value appeared high, because the mean ΔI for the negative control was low. The
relative standard error of the mean for the 10000pM, 100pM, and 1pM samples were 7%, 11%,
and 14%, respectively. This suggested that these trials had high repeatability.
In the trials using probe 2A, the mean ΔI for the 100mM Neg Ctrl, the 10000pM, 100pM, and
1pM samples were 0.284nA, 3.13nA, 2.04nA, and 0.643nA, respectively. The standard error of
the mean for the 100mM Neg Ctrl, the 10000pM, 100pM, and 1pM samples were 0.203nA,
0.215nA, 0.135nA, and 0.156nA, respectively. The data were statistically significant, since the
standard error bar of the negative control did not overlap with the ones from the trials that used
complementary DNA sequence (Figure 17). The precision of the assay was expressed via relative
standard error of the mean. The relative standard error of the mean for the negative control was
71%. This value appeared high, because the mean ΔI for the negative control was low. The
relative standard error of the mean for the 10000pM, 100pM, and 1pM samples were 7%, 7%,
and 24%, respectively. This suggested that these trials had high repeatability.
For both probes, the results showed that the ΔI from hybridizing to the non-complementary
sequence was lower than any of the ΔI from hybridizing to the complementary sequence (Figure
17). This demonstrated that both probes had a greater affinity for the complementary target than
the non-complementary target, even though the concentration of the non-complementary DNA
was greater than those of the complementary DNAs. Furthermore, a higher concentration of the
incubated complementary DNA resulted in a higher ΔI for both 1C and 2A probe assays (Figure
17). This provided further evidence that the probes were specific, since the ΔI depended on the
target concentration. The results also showed that the limit of detection of the assays were 1pM
of DNA (Figure 17). It was interesting to note that the ΔI from the hybridization of the probe 1C
to its complementary targets was greater than the corresponding ΔI using the probe 2A for all 3
target concentrations (Figure 17). In addition, the ΔI from the hybridization of the probe 1C to
the non-complementary target was smaller than the corresponding ΔI using the probe 2A (Figure
17). This could be caused by differences in the qualities or binding affinities of the probes.
38
Figure 17: The detection of DNA targets with the NME biosensing assay.
A range of concentrations of the 22-nucleotide complementary DNA targets are used to
examine the limit of detection. The negative control is a 22-nucleotide non-complementary
DNA sequence. A) Probe 1C is hybridized to the complementary or non-complementary
DNA. B) Probe 2A is hybridized to the complementary or non-complementary DNA. For
both probes, the results show that the ΔI from hybridizing to the non-complementary
sequence is lower than any of the ΔI from hybridizing to the complementary sequence. This
demonstrates that both probes have a greater affinity for the complementary target than
the non-complementary target, even though the concentration of the non-complementary
DNA is greater than that of the complementary DNAs. Furthermore, a higher
concentration of the incubated complementary DNA results in a higher ΔI for both 1C and
2A probe assays. This provides further evidence that the probes are specific, since the ΔI
depends on the target concentration. The results also show that the assays are capable of
detecting 1pM of DNA.
0.00
1.00
2.00
3.00
4.00
100nM Neg Ctrl 10000pM 100pM 1pM
ΔI (
nA
)
DNA Target Concentration
0.00
1.00
2.00
3.00
4.00
100nM Neg Ctrl 10000pM 100pM 1pM
ΔI (
nA
)
DNA Target Concentration
A
B
39
8.3 In vitro Transcribed RNA Target Biosensing
The NME biosensing assay was conducted with ~1000-nucleotide RNA sequences produced
from in vitro transcription, since influenza genome segment 7 RNA and matrix transcript RNA
were both ~1000-nucleotides in length1. This experiment was performed to determine if the
NME biosensing assay was capable of detecting long RNA molecules. In addition, several
concentrations of the RNA molecules were assayed to determine the sensitivity of the assay.
Both probes 1C and 2A were examined with the biosensing assay. The negative control was total
RNA from human lung epithelial cells, since the major nucleic acid contaminants from influenza
patient bronchial alveolar lavage samples were from human cells (Figure 18)26
. Since the
negative control contained a mixture of different RNA sequences, its concentration was
expressed in µg/µl rather than M (Figure 18). The 10000pM, 100pM, and 1pM concentrations of
the ~1000-nucleotide complementary RNA fragments were assayed (Figure 18). The negative
control target RNA was at least 25 times more concentrated than any of the complementary
targets (Figure 18). In this experiment, 10 independent trials were performed for each test
condition.
In the trials using probe 1C, the mean ΔI for the Neg Ctrl, the 10000pM, 100pM, and 1pM
samples were 0.203nA, 1.35nA, 0.813nA, and 0.379nA, respectively. The standard error of the
mean for the Neg Ctrl, the 10000pM, 100pM, and 1pM samples were 0.0263nA, 0.0516nA,
0.0756nA, and 0.0621nA, respectively. The data were statistically significant, since the standard
error bar of the negative control did not overlap with the ones from the trials that used
complementary RNA (Figure 18). The precision of the assay was expressed via relative standard
error of the mean. The relative standard error of the mean for the Neg Ctrl, 10000pM, 100pM,
and 1pM samples were 13%, 4%, 9%, and 16%, respectively. This suggested that these trials had
high repeatability.
40
Figure 18: The detection of RNA targets with the NME biosensing assay.
The positive samples are ~1000-nucleotide RNA produced from in vitro transcription, and
then serially diluted. The negative control is purified lung epithelial cell total RNA. A)
Probe 1C is hybridized to either the negative control or the influenza genome segment 7
RNA. B) Probe 2A is hybridized to either the negative control or the influenza matrix
transcript RNA. The ΔI resulting from the hybridization of the probe to the human RNA
(negative control) is less than the ΔI resulting from the hybridization of the probe to the
complementary RNA, even though the concentration of the human RNA is much greater
than that of the complementary RNA. In addition, the ΔI from the hybridization of the
probe to the complementary RNA is dependent on the RNA concentration. These trends
are observed for both probe 1C and 2A assays. These results show that the NME
biosensing assay is capable of differentiating large RNA molecules with complementary
sequences from a mixture of non-complementary RNAs. In addition, the results show that
the assays are capable of detecting 1pM of RNA.
0.00
0.40
0.80
1.20
1.60
1µg/µl Neg Ctrl 10000pM(40000pg/µl)
100pM(400pg/µl)
1 pM (4pg/µl)
ΔI(
nA
)
RNA Sample Concentration
0.00
0.40
0.80
1.20
1.60
1µg/µl Neg Ctrl 10000pM(40000pg/µl)
100pM(400pg/µl)
1 pM (4pg/µl)
ΔI (
nA
)
RNA Sample Concentration
B
A
41
In the trials using probe 2A, the mean ΔI for the Neg Ctrl, the 10000pM, 100pM, and 1pM
samples were 0.266nA, 1.19nA, 0.716nA, and 0.461nA, respectively. The standard error of the
mean for the Neg Ctrl, the 10000pM, 100pM, and 1pM samples were 0.0330nA, 0.0708nA,
0.0864nA, and 0.0562nA, respectively. The data were statistically significant, since the standard
error bar of the negative control did not overlap with the ones from the trials that used
complementary RNA (Figure 18). The precision of the assay was expressed via relative standard
error of the mean. The relative standard error of the mean for the Neg Ctrl, 10000pM, 100pM,
and 1pM samples were 12%, 6%, 12%, and 12%, respectively. This suggested that these trials
had high repeatability.
Similar to the experiment using the DNA target, the ΔI resulting from the hybridization of the
probe to the human RNA (negative control) was less than the ΔI resulting from the hybridization
of the probe to the complementary RNA, even though the concentration of the human RNA was
much greater than those of the complementary RNA (Figure 18). In addition, the ΔI from the
hybridization of the probe to the complementary RNA was dependent on the RNA concentration,
similar to the results from using DNA targets (Figure 18). These trends were observed for both
probe 1C and 2A assays. These results showed that the NME biosensing assay was capable of
differentiating large RNA molecules with the complementary sequence from a mixture of non-
complementary RNAs. In addition, the results showed that the assays were capable of detecting
1pM of RNA (Figure 18). Similar to the experiments that used DNA targets, the ΔI produced
when using the probe 1C was greater than the ΔI produced when using the probe 2A for
complementary targets, and lower from non-complementary targets (Figure 18). In general, the
hybridization of the probe to the RNA targets produced smaller ΔI than the hybridization of the
probe to the DNA targets (Figure 17, 18). This could be caused by the slower diffusion rate of
the large RNA molecules compared to the small DNA molecules, since small molecules diffuse
faster than large ones.
8.4 Cell Lysate Biosensing
Bronchial alveolar lavage was the main method to obtain samples from influenza patients26
.
Typically, these samples contained approximately 105 cells/ml and 10
9 viral particles/ml
26,28.
Transfected cells rather than patient samples were assayed as a safe non-biohazardous
alternative. To maintain a constant background cellular RNA concentration in all samples, all
42
cells were dilute to a constant cellular concentration before they were lysed. Moreover, the
lysates from the non-transfected cells were used as the diluent to dilute the influenza RNA in the
transfected cells. The diluted lysates of the transfected cells were hybridized to the probes to
determine if the NME biosensing assay could detect the viral RNA present within a mixture of
cellular nucleic acids and cell debris.
Figure 19: The detection of influenza RNA targets from cell lysates using the NME
biosensing assay.
Lung epithelial cells are transfected with plasmids to produce either the influenza segment
7 genomic RNA or the influenza matrix transcript RNA. The concentration of lysed cells is
chosen to reflect the number of cells in bronchial alveolar lavage. The influenza RNA
concentration is chosen to reflect the viral load in infected patients. The negative control is
un-transfected cell lysates. For both probe assays, the results show that the ΔI from the
transfected cell samples is higher than the ΔI from the non-transfected cell samples. This
demonstrates that the assay is capable of differentiating between samples with viral RNAs
from the samples without, at clinically relevant viral RNA and cellular RNA
concentrations. In addition, the results show that the assay is capable of detecting influenza
RNA in complex biological samples.
In this experiment, 10 independent trials were performed for each test condition. The mean ΔI
for the 2A probe 50cells/µl (negative control) sample, the 2A probe 50cells/µl 106 viral RNA/µl
sample, the 1C probe 50cells/µl (negative control) sample, and the 1C probe 50cells/µl 106 viral
RNA/µl sample were 0.278nA, 0.596nA, 0.407nA, and 0.836nA, respectively. The standard
error of the mean for the 2A probe 50cells/µl (negative control) sample, the 2A probe 50cells/µl
106 viral RNA/µl sample, the 1C probe 50cells/µl (negative control) sample, and the 1C probe
50cells/µl 106 viral RNA/µl sample were 0.0940nA, 0.0699nA, 0.0808nA, and 0.0444nA,
0.00
0.20
0.40
0.60
0.80
1.00
2A Probe; 50Cells/µl
2A Probe; 50Cells/µl; 1E+6
Viral TranscriptRNA/µl
1C Probe; 50Cells/µl
1C Probe; 50Cells/µl; 1E+6Viral Genomic
RNA/µl
ΔI(
nA
)
Probe Name and Sample Composition
43
respectively. The data were statistically significant, since the standard error bar of the negative
control did not overlap with the ones from the trials that used viral RNA (Figure 19). The
precision of the assay was expressed via relative standard error of the mean. The relative
standard error of the mean for the 2A probe 50cells/µl (negative control) sample, the 2A probe
50cells/µl 106 viral RNA/µl sample, the 1C probe 50cells/µl (negative control) sample, and the
1C probe 50cells/µl 106 viral RNA/µl sample were 34%, 12%, 20%, and 5%, respectively. This
suggested that these trials had high repeatability.
For both probe assays, the results showed that the ΔI from the transfected cell samples was
higher than the ΔI from the non-transfected cell samples (Figure 19). This demonstrated that the
assay was capable of differentiating the samples that contained viral RNA from the samples that
did not, at clinically relevant viral RNA and cellular RNA concentrations. In addition, the results
showed that the assay was capable of detecting influenza RNA in complex biological samples
(Figure 19). Similar to the experiments that used DNA targets or in vitro transcribed RNA
targets, the ΔI produced when using the probe 1C was greater than the ΔI produced when using
probe 2A (Figure 19).
8.5 RNA Quantitation
QPCR was performed on the in vitro transcribed RNA and the RNA from the lysed transfected
cells. QPCR was used to ensure that the correct sequence was produced from in vitro
transcription. In addition, it was used to create a standard curve with the RNA from in vitro
transcription and to quantitate the influenza RNA produced from the transfected cells.
The primers used for the QPCR were the universal primers designed by CDC to detect H1N1
influenza A27
. The primers were designed to amplify a region on the influenza matrix gene,
located on genome segment 7. They were capable of amplifying both the genomic RNA and the
transcript RNA. The standard curve was constructed with in vitro transcribed RNA in tenfold
dilution intervals from 104 to 10
10 copies/µl (Figure 20). The concentration of the genomic RNA
from cell transfection was determined to be 15.1*106 copies/µl (Figure 20). The concentration of
the transcript RNA from cell transfection was determined to be 4.41*106 copies/µl (Figure 20).
44
Figure 20: The determination of influenza RNA concentration from lysate using QPCR.
The in vitro transcribed RNAs are quantified with the Nanodrop 2000 UV-Vis
spectrophotometer, and plotted as the triangle data points to construct the standard curves.
The concentrations of the influenza RNAs from the lysate are calculated from the standard
curve and plotted as the square data points. A) The concentration of segment 7 genomic
RNA that binds to probe 1C is 1.517 copies/µl. B) The concentration of matrix RNA
transcript that binds to probe 2A is 4.416 copies/ µl.
1.51E+07
0
5
10
15
20
25
30
1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08 1.0E+09 1.0E+10
Thre
sho
ld C
ycle
RNA Concentration (Copies / µl)
A
4.41E+06
0
5
10
15
20
25
30
1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08 1.0E+09 1.0E+10
Thre
sho
ld C
ycle
RNA Concentration (Copies / µl)
B
45
Chapter 4 Conclusion and Future Directions
9 Conclusion
A diagnostic assay based on the NME biosensing assay was developed to detect both influenza A
transcript RNA and influenza A genomic RNA. The development of this assay required the
construction of highly specific probe sequences, the selection of defect free and contaminant free
chips, the purification of probes capable of binding specifically to complementary sequences,
and the optimization of the probe and the MCH concentrations for deposition.
The assay was capable of differentiating 1pM of complementary DNA from 100nM of non-
complementary DNA. In addition, it was able to differentiate 4pg/µl of large complementary
RNA molecules from 1µg/µl of non-complementary mixed RNA molecules. Finally, the assay
was able to detect 106 copies/µl of viral RNA in a complex solution containing 50cell/µl of lysed
cells. The development and optimization of this assay was a significant step toward the rapid
detection of influenza particles in a clinical setting.
10 Future Directions
The progress made in developing an influenza diagnostic assay can be taken further by
improving the assay parameters and by expanding the assay applications. Several assay
parameters can be improved so that the assay can be performed in the clinical setting. This
requires further characterizing and optimizing the assay conditions, stabilizing the probe and
NME for storage, and testing patient samples. The applications of the assay can be expanded to
detect more than just the conserved influenza A sequence. This requires designing and testing
new probes from other influenza strains and other viruses, and designing a multiplexed platform.
Ideally, the only steps required to use the diagnostic device in the clinic are reading the
background signal, applying the sample, incubating the sample, and reading the sample signal.
However, several challenges need to be resolved. A major challenge is the susceptibility of the
probe, the NME, and the scanning solution to degradation. Due to this susceptibility, the plating
of the NME, the deposition of the sample, and the preparation of the scanning solution must be
performed immediately prior to assaying the sample. Thus, this prevents the creation of chips
46
with pre-deposited probes. To overcome these issues, a case needs to be designed to
mechanically protect the NME from damage and dust particles. In addition, the PNA probes need
to be stabilized from chemical and biological degradation. Finally, an airtight container is
required to store the scanning solution.
Another challenge is the strict reliance on specific assay conditions, where any deviation from
these conditions can cause false positives or false negatives. Experiments need to be performed
to determine how different parameters affect the assay. The parameters relating to probe design
and synthesis include probe length, probe GC content, probe synthesis conditions, and HPLC
purification conditions. The parameters relating to NME formation include chip preparation
process, NME size, NME nanostructuring, and NME composition. The parameters relating to
deposition conditions include acetonitrile concentration, probe:MCH ratio, probe concentration,
deposition time, and deposition temperature. The parameters relating to electrochemical
measurements include scanning solution, incubation time, incubation temperature, and sample
preparation. An understanding of how each parameter affects the assay allows for the
optimization of these parameters.
A third challenge that prevents the assay from being clinic ready is the possibility that the patient
samples contain factors that can interfere with the assay. The ability of the assay to detect
influenza in bronchial alveolar lavage samples from flu patients needs to be tested. For this
experiment, the negative control is bronchial alveolar lavage from healthy samples, and the
positive control is bronchial alveolar lavage from healthy samples spiked with influenza
particles. Samples from patients infected with influenza, patients with other viral respiratory
infections, patients with bacterial respiratory infections, and healthy patients need to be
examined.
The accuracy of influenza diagnosis can be improved by assaying the same patient sample with
multiple different probes. Potential relevant probes include sequences conserved in specific
subtypes of influenza A viruses, and sequences from influenza B and influenza C. Moreover,
conserved sequences from other respiratory viruses such as the adenovirus can be used to
provide a more detailed diagnosis on the cause of flu-like symptoms.
To limit the cost and time required for the assay and to prevent the degradation of the patient
samples, assaying with multiple probes requires a multiplexed platform. This platform requires
47
the design of a single chip that contains multiple NMEs, so that one chip is sufficient to examine
multiple sequences from the same patient sample. In addition to a multiplexed chip design, a
process to deposit different probes in close proximity needs to be developed. The chip also needs
to be compartmentalized to run the negative control samples in parallel with the patient samples.
Moreover, hardware needs to be developed to connect the multiplexed chip to the potentiostat.
Finally, software needs to be programed to integrate the data collected from the multiplexed
chip.
48
References
1 Nicholson, K. G., Wood, J. M. & Zambon, M. Influenza. The Lancet 362, 1733-1745
(2003).
2 Hampson, A. W. & Mackenzie, J. S. The Influenza Viruses. (Australasian Medical
Publishing Company, 2006).
3 Low, D. Reducing Antibiotic Use in Influenza: Challenges and Rewards. Clinical
Microbiology and Infection 14, 298-306 (2008).
4 Takahashi, H., Otsuka, Y. & Patterson, B. K. DiagnosticTests for Influenza and Other
Respiratory Viruses: Determining Performance Specifications Based on Clinical Setting.
Journal of Infection & Chemotherapy 16, 155-161 (2010).
5 Drummond, T. G., Hill, M. G. & Barton, J. K. Electrochemical DNA Sensors. Nat
Biotech 21, 1192-1199 (2003).
6 Fang, Z. & Kelley, S. O. Direct Electrocatalytic mRNA Detection Using PNA-Nanowire
Sensors. Analytical Chemistry 81, 612-617 (2009).
7 Petric, M., Comanor, L. & Petti, C. A. Role of the Laboratory in Diagnosis of Influenza
During Seasonal Epidemics and Potential Pandemics. The Journal of infectious diseases
194 S98-110 (2006).
8 Khanna, M., Kumar, P., Choudhary, K., Kumar, B. & Vijayan, V. Emerging Influenza
Virus: A Global Threat. Journal of Biosciences 33, 475-482 (2008).
9 Amano, Y. & Cheng, Q. Detection of Influenza Virus: Traditional Approaches and
Development of Biosensors. Analytical and Bioanalytical Chemistry 381, 156-164
(2005).
10 Webby, R. J. & Webster, R. G. Are We Ready for Pandemic Influenza? Science 302,
1519-1522 (2003).
11 Monto, A. S. Epidemiology of Influenza. Vaccine 26, D45-D48 (2008).
12 Beveridge, W. I. The Chronicle of Influenza Epidemics. Vol. 13 (1991).
13 Hampson, A. W. in Perspectives in Medical Virology Vol. 7 (ed C. W. Potter) 49-85
(Elsevier, 2002).
14 Farias, J. A. et al. Critically Ill Infants and Children with Influenza A (H1N1) in Pediatric
Intensive Care Units in Argentina. Intensive Care Medicine 36, 1015-1022 (2010).
15 Cutter, J. L. et al. Outbreak of Pandemic Influenza A (H1N1-2009) in Singapore, May to
September 2009. Annals of the Academy of Medicine, Singapore 39, 273-210 (2010).
49
16 Sakai-Tagawa, Y. et al. Sensitivity of Influenza Rapid Diagnostic Tests to H5N1 and
2009 Pandemic H1N1 Viruses. Journal of Clinical Microbiology 48, 2872-2877 (2010).
17 Levy, S. B. & Marshall, B. Antibacterial Resistance Worldwide: Causes, Challenges and
Responses. Nat Med (2004).
18 Leland, D. S. & Ginocchio, C. C. Role of Cell Culture for Virus Detection in the Age of
Technology. Clinical Microbiology Reviews 20, 49-78 (2007).
19 Dwyer, D. E., Smith, D. W., Catton, M. G. & Barr, L. G. Laboratory Diagnosis of
Human Seasonal and Pandemic Influenza Virus Infection. (Australasian Medical
Publishing Company, 2006).
20 Ellis, J. S. & Zambon, M. C. Molecular Diagnosis of Influenza. Vol. 12 (Wiley, 2002).
21 Charlton, B., Crossley, B. & Hietala, S. Conventional and Future Diagnostics for Avian
Influenza. Comparative Immunology, Microbiology and Infectious Diseases 32, 341-350
(2009).
22 Soleymani, L., Fang, Z., Sargent, E. H. & Kelley, S. O. Programming the Detection
Limits of Biosensors Through Controlled Nanostructuring. Nature Nanotechnology 4,
844-848 (2009).
23 Lapierre-Devlin, M. A. et al. Amplified Electrocatalysis at DNA-Modified Nanowires.
Nano Letters 5, 1051-1055 (2005).
24 Lam, B., Fang, Z., Sargent, E. H. & Kelley, S. O. Polymerase Chain Reaction-Free,
Sample-to-Answer Bacterial Detection in 30 Minutes with Integrated Cell Lysis.
Analytical Chemistry 84, 21-25 (2011).
25 Soleymani, L. et al. Hierarchical Nanotextured Microelectrodes Overcome the Molecular
Transport Barrier To Achieve Rapid, Direct Bacterial Detection. Acs Nano 5, 3360-3366
(2011).
26 Domagała, K. J., Skirecki, T., Maskey, W. M., Grubek, J. H. & Chazan, R.
Bronchoalveolar Lavage Total Cell Count in Interstitial Lung Diseases—Does It Matter?
Inflammation 35, 803-809 (2012).
27 Global-Alert-Response. Pandemic (H1N1) 2009 Guidance Documents (GAR
Publications, World Health Organization, 2009).
28 To, K. W. et al. Viral Load in Patients Infected with Pandemic H1N1 2009 Influenza A
Virus. Journal of Medical Virology 82, 1-7 (2010).
50
Appendix I: Mass Spectrometry Spectrum
Mass Spectrometry Spectrum for Probe 2A
+TOF MS: 23 MCA scans from 20120420_015 Sean 2A36.wiffa=3.56598087820512760e-004, t0=5.98822642784070920e+001
Max. 6783.0 counts.
1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500m/z, amu
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
258
Inte
ns
ity
, c
ou
nts
4050.6079
6435.5990
3585.40954580.8735
4855.8967
5107.0380
1747.7349 4314.77853226.3208
3517.4684
1581.6550
4117.6727 6483.2112882.5553
5982.90983782.56312131.90771039.4493
2407.03105398.1079
4382.5819 6589.61421963.85441456.6105
+TOF MS: 48 MCA scans from 20120420_016 Sean 2A38.wiffa=3.56598087820512760e-004, t0=5.98822642784070920e+001
Max. 1083.0 counts.
1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4m/z, amu
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1083
Inte
ns
ity
, c
ou
nts
679.5042
4580.8967
4855.96234049.6546
4314.78425107.0520
5400.5385
3585.39276435.5860882.5419
3851.51204118.60733516.4585
637.2941 1421.6257 3226.3212 5693.99722131.9148 4382.6890 5981.1443
2409.01141039.44982291.7706 5251.00624470.7267 6588.4689 9441.0871 9966.7960 11292.5836
51
Mass Spectrometry Spectrum for Probe 1C
+TOF MS: 49 MCA scans from 20120508_011 Sean 1C40.wiffa=3.56609674488498070e-004, t0=5.87080628066032660e+001
Max. 995.0 counts.
1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4m/z, amu
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
995
Inte
ns
ity
, c
ou
nts
4582.3320
4855.8624
4316.66904050.5818
5400.69775106.8456
5691.7759
3585.40764117.4871 6435.6279
5983.43514053.4862637.3087 3852.3909
4385.47946485.18533226.3316
2409.0212
2133.8936
6590.38244388.5676
2292.3604 4693.33262024.8851 9966.97905543.1649882.5457 2659.0764 6259.9748 9439.79674203.6098 8633.9514
+TOF MS: 74 MCA scans from 20120508_012 Sean 1C42.wiffa=3.56609674488498070e-004, t0=5.87080628066032660e+001
Max. 822.0 counts.
1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4m/z, amu
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
822
Inte
ns
ity
, c
ou
nts
4858.2984
4580.7760
5112.0524
5400.5981
5692.0802
4048.65964314.6879 6436.1875637.3049
5983.3945
3517.3833 6485.3696
3584.3577 4116.6489
2131.93843850.5092
6588.4960
2408.9959
5000.2246659.2884 6602.07105246.96964722.2938
2291.9593 9966.84746324.2765 11215.17632025.5938 9717.46113939.6524 6122.53375875.5202
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