Utilization of ferrioxamine microarrays for the rapid detection of pathogenic bacteria

123 Pages·2014·4.07 MB·English

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12-2016
Utilization of ferrioxamine microarrays for the rapid
detection of pathogenic bacteria
Nigam Bir Arora
Purdue University
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Arora, Nigam Bir, "Utilization of ferrioxamine microarrays for the rapid detection of pathogenic bacteria" (2016).Open Access
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https://docs.lib.purdue.edu/open_access_dissertations/903
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PURDUE UNIVERSITY
GRADUATE SCHOOL
Thesis/Dissertation Acceptance
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NigamBirAroraII
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(cid:3) PATHOGENICBACTERIA
DoctorofPhilosophy
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UTILIZATION OF FERRIOXAMINE MICROARRAYS FOR THE RAPID
DETECTION OF PATHOGENIC BACTERIA
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Nigam Bir Arora II

In Partial Fulfillment of the
Requirements of the Degree
of
Doctor of Philosophy

December 2016
Purdue University
West Lafayette, Indiana
ii

I dedicate this work to my family; to my father for instilling in me the importance of
education and the drive to pursue knowledge, to my mother for providing undying
support throughout my education and to my sister for leading the way ever since I can
remember.
iii

ACKNOWLEDGMENTS
I would like to acknowledge the educators that played a part in helping me reach
this point. First is Professor Stephen Randall who gave me an opportunity to join his lab
during my undergraduate education at IUPUI. This is where I got my first taste of
laboratory research. Next is Professor Alex Wei who accepted me as a PhD student in
his lab in 2012. Since then I have developed greatly as a scientist, a speaker and most
importantly as an analytical thinker. I am very appreciative of both these opportunities
and the knowledge and experience I have gained along the way. I would also like to
thank some of my fellow students who helped me along the way throughout my
education. Dr. Gauge Koelher was the first graduate student that I grew to know well.
During my time in the Randall lab she never turned down a question and helped me early
on establish my foothold in basic laboratory techniques. Dr. Qingshan Wei, the graduate
student I worked with during my initial rotation in the Wei lab, introduced me to a whole
new side of research related to novel chemistry and fascinating nanoparticle based
studies. My experience working with him was a major influence in my decision to join
the Wei lab for the remainder of my PhD. Dr. Matt Casselman and Dr. Oscar Morales
were the two most senior graduate students when I joined the Wei lab. They took me in
and each did their fair share in mentoring me through my early days in the lab. I would
also like to thank Karl Williams and his family for taking me into their family home
iv
during my final semester at Purdue. Finally I would like to thank my sister Dr. Natasha
Arora. She, more than any other person, has offered me support when I needed it most to
keep going throughout my academic career.
v

TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ......................................................................................................... viii
LIST OF SCHEMES......................................................................................................... xii
ABSTRACT ..................................................................................................................... xiii
CHAPTER 1. SIDEROPHORES AS IMMUTABLE LIGANDS ......................................1
1.1. Introduction to Siderophores...................................................................................1
1.2.Immutable Ligands for Bacterial Detection .............................................................3
1.2.1. Methods of Pathogen Detection and Associated Pitfalls ...............................4
1.3.Ferrioxamine as an Immutable Ligand for Pathogen Detection ..............................5
1.4. Bacterial Ferrioxamine Receptors Found in Literature ..........................................6
1.5. Protein BLAST Search and Analysis ......................................................................8
1.6. Conclusions ...........................................................................................................10
1.7. Materials and Methods ..........................................................................................11
1.8. References .............................................................................................................13
CHAPTER 2. OPTIMIZATION OF MICROARRAYS FOR DETECTION OF
FERRIOXAMINE BINDING PATHOGENS ...................................................................19
2.1. Introduction to Microarrays for Detection of Pathogenic Bacteria ......................19
2.2. Chip Preparation and Optimization ......................................................................20
2.2.1. Synthesis of FITC Conjugates for Optimization of Chip Preparation ........21
2.2.1.1. FOx–BSA Conjugates and Associated Pitfalls ...................................22
2.2.1.2. Synthesis of Bis-isothiocyanate (ITC) Linkers ...................................24
2.2.1.3. Synthesis of Low-Molecular Weight FOx Conjugates .......................26
2.2.1. Optimization of Chip Preparation ...............................................................29
vi
Page

2.2.2.1. Synthesis of FITC Conjugate for Optimization of Chip
Preparation .......................................................................................................29
2.2.2.2. Optimization of pH and Humidity Conditions with FITC
Conjugate .........................................................................................................30
2.2.2.3. Minimization of Background Noise....................................................32
2.3. Culturing of Pathogenic Bacteria in Iron-Restricted Media .................................34
2.4. Pathogen Detection ...............................................................................................36
2.5. Initial Use of Small-Molecule Conjugates for Screening Bacteria .......................37
2.6. Limit of Detection Using Ferrioxamine Microarrays ...........................................40
2.7. Optimization of PathoTest Device (MarkV) .........................................................43
2.8. Conclusions ...........................................................................................................46
2.10. Materials and Methods ........................................................................................46
2.11. References ...........................................................................................................55

CHAPTER 3. ULTRA RAPID DETECTION OF BACTERIAL PATHOGENS ............57
3.1. Introduction to Rapid Pathogen Detection ............................................................57
3.2. Synthesis of Ferrioxamine Conjugates .................................................................60
3.3. Preliminary Studies Utilizing Drop-on-Chip Method ...........................................62
3.4. Pathogen Detection Using Horizontal Submersion Method .................................67
3.5. Ultra-rapid Detection of Y. enterocolitica, S. aureus, S. enterica and A.
baumannii ..............................................................................................................69
3.6. Detection of Y. enterocolitica Cultured at 4 °C ....................................................80
3.7. Conclusions ...........................................................................................................81
3.8. Materials and Methods ..........................................................................................82
3.9. References .............................................................................................................89
VITA ..................................................................................................................................94
PUBLICATION .................................................................................................................95
vii
LIST OF TABLES
Table Page
1.1 Bacterial strains subjected to BLAST search based on expression of FOx receptors .. 9
2.1 List of all bacterial strains optimized for growth under iron restricted conditions ..... 35
3.1 Summary of S/N values from bacterial detection studies using the drop-on-chip
method......................................................................................................................... 64
3.2 Summary of time-to-detection experiments utilizing the horizontal submersion
method, with mean S/N values from three independent FFT analyses ....................... 72
3.3 Summary of time-to-detection experiments utilizing the horizontal submersion
method, with mean S/B values from three independent FFT analyses ....................... 73