Download Microfluidic based Sample Preparation for Bloodstream Infections

Microfluidic based Sample Preparation for
Bloodstream Infections
Sahar Ardabili
Kungliga Tekniska högskolan, KTH
Royal Institute of Technology
School of Biotechnology
Stockholm, 2014
© Sahar Ardabili
Stockholm, 2014
Royal Institute of Technology
Science for Life Laboratories
SE-171 65 Solna
Printed by Universitetservice US-AB
Drottning Kristinas väg 53B
SE-100 44 Stockholm
Sweden
ISBN 978-91-7595-385-4
TRITA-BIO Report 2014:19
ISSN 1654-2312
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To my parents
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Abstract
Microfluidics promises to re-shape the current health-care
system by transferring diagnostic tools from central laboratories to close
vicinity of the patient (point-of-care). One of the most important
operational steps in any diagnostic platform is sample preparation, which
is the main subject in this thesis. The goal of sample preparation is to
isolate targets of interest from their surroundings. The work in this thesis
is based on three ways to isolate bacteria: immune-based isolation,
selective cell lysis, size-based separation.
The first sample-preparation approach uses antibodies against
lipopolysaccharides (LPS), which are surface molecules found on all
gram-negative bacteria. There are two characteristics that make this
surface molecule interesting. First, it is highly abundant: one bacterium
has approximately a million LPS molecules on its cell-wall. Second, the
molecule has a conserved region within all gram-negative bacteria, so
using one affinity molecule to isolate disease-causing gram-negative
bacteria is an attractive option, particularly from the point of view of
sample preparation. The main challenge, however, is antigen
accessibility. To address this, we have developed a treatment protocol
that improves the capturing efficiency.
The strategy behind selective cell lysis takes advantage of the
differences between the blood-cell membrane and the bacterial cell-wall.
These fundamental differences make it possible to lyse (destroy) bloodcells selectively while keeping the target of interest, here the bacteria,
intact and, what is more important alive. Viability plays an important role
in determining antibiotic susceptibility.
Difference in size is another well-used characteristic for sampleseparation. Inertial microfluidics can focus size-dependent particle at
high flow-rates. Thus, particles of 10 µm diameter were positioned in
precise streamlines within a curved channel. The focused particles can
then be collected at defined outlets. This approach was then used to
isolate white blood cells, which account for approximately 1% of the
whole blood. In such a device particles of 2µm diameter (size of bacteria)
would not be focused and thereby present at every outlet. To separate
bacteria from blood elasto-inertial microfluidics was used. Here, e blood
components are diverted to center of the channels while smaller bacteria
remain in the side streams and can subsequently be separated
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Populärvetenskaplig sammanfattning
Blodförgiftning (sepsis) är en livshotande sjukdom som årligen
drabbar omkring 15-19 millioner människor globalt[1–3]. Den
bakomliggande anledningen är oftast en bakterieinfektion, men
blodförgiftning kan även orsakas av virus, parasiter eller
svampinfektioner[4]. I Sverige är blodförgiftning den 13:e mest
resurskrävande sjukdomen inom slutenvården[5]. Sepsis är en följd av
en så kallad systemisk inflammationsrespons (SIRS) orsakat av vårt eget
immunförsvar som svar på invaderande patogener (sjukdomsorsakande
organismer). Detta är ett exempel på hur vårt immunsystem, som i
vanliga fall ska skydda oss från faroämnen, kan orsaka mer skada än
nytta. Om sjukdomen lämnas obehandlad kan det på sikt leda till
cirkulationskollaps,
multiorgansvikt
och
död[6].
Ett
snabb
omhändertagande med rätt antibiotikabehandling räddar liv. Därför ges
en kombination av olika antibiotika omgående om en patient misstänks
ha drabbats av sepsis[6]. Idag används tekniken blododling för isolering
och identifiering av den invaderande bakterien, samt fastställande av
eventuell antibiotikaresistens. Nackdelen med denna teknik är att
svarsresultat kan dröja upp till 72 timmar och då är det ofta för sent[7–
11]. Läkare behandlar i blindo då situationen lätt kan bli livshotande.
Behovet för en diagnostisk plattform med snabb patogenidentifiering är
därmed stort.
Målet med studierna som föregått denna avhandling är provpreparing med hjälp av mikrofluidik. Mikrofluidik är ett interdisciplinärt
forskningsfält där mikrosystemteknik, fysik och bioteknik möts för att
skapa ett system i miniatyr där diverse laboratorieanalyser kan utföras.
Ett stort fokus har lagts på bakterieisolering med förhoppningen om att
detta ska ta oss ett steg närmare förbättrade diagnostiska verktyg för
sjukdomar som sepsis.
Avhandlingen är uppdelad i 4 kapitel. Kapitel 1 ger en
sammanställning över motiveringen bakom denna forskning samtidigt
som den beskriver de diagnostiska verktyg som finns tillgängliga på
marknaden idag. Kapitel 2 redogör för framstegen som gjorts inom
mikrofluidik för att isolera celler från komplexa lösningar så som blod.
Kapitel 3 beskriver det eftersträvade slutmålet med forskningen, och
beskriver koncepten ”point-of-care” och ”lab-on-a-chip”. Slutligen
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redogör Kapitel 4 för den bakomliggande forskningen som jag har utfört
tillsammans med mina kollegor under min forskningsutbildning.
Avslutningsvis skulle jag vilja poängtera att bakterier inte är ondskan
inkarnerad.
Vår kropp innehåller ungefär 10 gånger så många
bakterieceller än av våra egna celler[12]. Dessa bakterier hjälper oss på
olika sätt, t.ex. med matsmältning, vitaminproduktion, och
immunförsvar[12]. Vår kropp kommer bara till skada när de hamnar vid
fel plats.
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Numinous - English, (adj) - "describing an experience that makes you
fearful yet fascinated, awed yet attracted; the powerful, personal feeling
of being overwhelmed and inspired.
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List of publications
Ardabili, S., Zelenin, S., Ramachandraiah, H., Russom, A. Epitope
unmasking for improved immuno-magnetic isolation of Gram-negative
bacteria. Manuscript
Zelenin, S., Hansson, J., Ardabili, S., Ramachandraiah, H., Brismar, H.,
and Russom, A. Microfluidic-based isolation of bacteria from whole
blood for sepsis diagnostics. Biotechnology Letters, 2014, DOI:
10.1007/s10529-014-1734-8
Ramachandraiah, H.*, Ardabili, S.*, Faridi, A. M., Gantelius, J.,
Kowalewski, J. M., Mårtensson, G., & Russom, A. Dean flow-coupled
inertial focusing in curved channels. Biomicrofluidics, 2014, 8(3),
034117.
Faridi, A.M., Ramachandraiah, H., Ardabili, S., Zelenin, S., and Aman
Russom, Elasto-Inertial microfluidics for bacteria separation from whole
blood for sepsis diagnostics. Manuscript
Pavankumar, A.S.*, Ardabili, S*, Zelenin, S., Shulte, T., Lundin, A. and
Russom, A. Recombinant Shigella flexneri apyrase enzyme for
bioluminescence based diagnostic applications Manuscript
* Authors contributed equally.
All papers reproduced with permission of the copyright holders.
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Contribution to the papers
Paper I:
Major parts of the experiments and writing.
Paper II:
Minor parts of the experiment and writing
Paper III:
Major parts of the experiments. Minor parts of the writing
Paper IV:
Minor parts of the experiments and writing
Paper V:
Major parts of the experiments. Minor parts of the writing
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TABLE OF CONTENT
Abstract .............................................................................................4
Populärvetenskaplig sammanfattning ......................... 5
List of publications ...................................................... 8
Contribution to the papers ................................................................9
Thesis Road Map ............................................................................. 12
Bloodstream infection ..................................................................... 13
Infectious disease ...................................................... 14
Sepsis ........................................................................ 16
Epidemiology .......................................................................... 17
Misdiagnosis: a fatal error. ..................................................... 18
Current diagnostic assays .......................................... 19
Nucleic acid-based techniques............................................... 20
Positive blood culture ............................................................. 21
Diagnosis directly from blood ................................................ 22
Isolation techniques for complex fluids .........................................25
The Challenge: Taking Blood Apart ........................... 26
Microfluidics – A Laboratory Time Saver? ................ 27
Microfluidic-Based Separation .................................. 30
Cell-Wall Composition ...............................................31
Chemical approach................................................................. 34
Affinity-Based Approaches .................................................... 34
Size-Based Approaches .......................................................... 36
Inertial Microfluidics ............................................................. 36
Point-of-Care: The Final Goal ......................................................... 37
Point-of-Care: An Overview ....................................... 38
Operational Steps within Point-of-Care ..................... 39
Point-of-Care for Bacterial Identification .................. 40
Verigene.................................................................................. 40
Film Array ............................................................................... 41
Concluding Remarks .............................................................. 42
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Present investigation ...................................................................... 43
Aim of the Thesis ....................................................... 44
Paper I ....................................................................... 46
Paper II……………………………………………………………….50
Paper III .................................................................... 54
Paper IV .................................................................... 57
Paper V ...................................................................... 60
Conclusion and Future Work ......................................................... 63
Acknowledgement .......................................................................... 66
Abbreviation ................................................................................... 69 11
Thesis Road Map
This thesis focuses on sample preparation with an emphasis on
microfluidics. The objective is to apply such strategies to the development
of diagnostics for infectious disease.
There are four chapters of which Chapters 1-3 are introductory.
Chapter 1 (Blood Stream Infection) gives an overview of the motivation
behind our research as well as a brief review of the diagnostic tools
available on the market today. Chapter 2 reviews the work in
microfluidics to isolate cells from complex fluids. Chapter 3 presents
the concept of point-of-care and lab-on-a-chip: the intention is, in
principle, to miniaturize a full-scale laboratory onto a tiny chip for
integrated bioassays. Finally Chapter 4 presents my work during my
years as a PhD student.
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CHAPTER 1
Bloodstream infection
“Our arsenals for fighting off bacteria are so powerful, and involve so
many different defense mechanisms, that we are in more danger from
them than from the invaders. We live in the midst of explosive devices;
we are mined. It is the information carried by the bacteria that we
cannot abide. The Gram-negative bacteria are the best examples of this.
They display lipopolysaccharide endotoxin in their walls, and these
macromolecules are read by our tissues as the very worst of bad news.
When we sense lipopolysaccharide, we are likely to turn on every defense
at our disposal; we will bomb, defoliate, blockade, seal off, and destroy all
the tissues in the area.
-
Thomas Lewis (The Lives of a Cell: Notes from a Biology
Watcher) [1]
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Infectious disease
The unwanted presence of multiplying pathogens in our bodies
can lead to a range of infectious diseases. The infection-causing
pathogens may be viruses, bacteria, fungi, protozoa, parasites or prions
[2]. Infectious disease affects a vast number of people world-wide.
According to a report from the World Health Organization in 2011,
infectious diseases such as lower respiratory infections, HIV/AIDS,
diarrheal diseases, malaria and tuberculosis are the leading causes of
death in low-income countries. In high-income countries, on the other
hand, only one in ten deaths are caused by infectious disease [3] (Figure
1.1).
Figure 1.1: Leading causes of deaths world-wide in 2011. Chart adapted from WHO- fact
sheet. [3].
Nevertheless, health-care associated infection in the intensive
care units (ICU) remains a global problem and is associated with high
mortality and costs [4–8]. The risk of acquiring an infection is large even
in high-income countries. An 2009 ICU study covering 75 countries
world-wide with data collected from 13,796 patients on one single day,
reported that 51% of all of the patients were considered infected [4].
Within the infected cohort, only 70% had a culture-positive result, but
antibiotics were administered to 71% of all the patients [4]. An earlier,
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one-day study from 1992 with data from 17 European countries showed a
similar percentage of infected patients (45%) [8].
It should not be surprising that the risk of acquiring an infection
in hospital settings is high, especially in view of increasing number of
entry points created by invasive procedures. Incorrect antibiotics for
bacterial infections can have dire life-threatening consequences [9]. The
number of patients receiving inappropriate antibiotics has been estimated
to be 20-30% [10]. The level of antibiotic resistance has steadily
increased over the years while the production of new effective antibiotics
has decreased. Together this constitutes an alarming scenario in a healthcare era in which diseases that could have been easily treated in the past
can now have mortal outcomes [11–13].
The yearly incidence of sepsis, the systemic inflammatory
response to an infection, is approximately 18 million people worldwide
[14–17]. This would correspond to the total population of Denmark,
Finland, Norway, and Slovenia World bank figures from 2012 [18].
According to the Society of Critical Care Medicine, sepsis is the second
leading cause of death in non-coronary ICUs in the USA [19–21].The
mortality rate of sepsis is estimated to be between 20% and 80% [14,21–
23]. These studies demonstrate the importance of infection control, which
can be achieved by improving hospital guidelines, providing new
medicines; developing better strategies for diagnostics and simply by
providing diagnostic tools applicable in resource limited areas.
In addition to clinical settings pathogen detection is of great
interest and importance in many fields, such as the food industry and in
water and environment health and safety (Figure 1.2). The present thesis
is primarily concerned with pathogen (bacteria) detection for clinical use.
Techniques for bacteria isolation (sample preparation) have been given
particular importance here.
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Figure 1.2: The relative amount of literature in specific areas. Chart adapted from Lazcka et
al 2007 [24].
Sepsis
Sepsis, severe sepsis and septic shock constitute thee systemic
inflammatory response syndrome (SIRS) of infectious origin with
escalating severity, symptoms and signs such as organ dysfunction and
hypotension [15,22,25–28]. The systemic inflammatory response is part
of the body’s defense mechanism against harmful invasions. However, in
the case of sepsis, severe sepsis and septic shock, this response has gone
awry and causes more harm than good. Infection is not the sole cause of
SIRS [25,26] (Figure 1.3). It can be triggered by a range of events such as
trauma, burns or pancreatitis. Additionally, Figure 1.3 sows that the
causative agent in sepsis is not always a bacteria but could also be fungi,
parasites or viruses [25]. Nonetheless, as the Figure 1.3 indicates, bacteria
(bacteremia) are the leading cause [21].
SIRS patients may display a range of different clinical
manifestations such as; fever (> 38°C), rapid heart rate (> 90 beats/min),
hyperventilation and changes in white blood-cell counts [22,25,28,29].
Two or more of these signs are needed to fulfill the criteria for SIRS. In
order to confirm that the underlying cause is actually an infection, further
investigation is required [28,29]. Because of the seriousness of the
condition, doctors administer broad-spectrum antibiotics immediately
when sepsis is suspected. There is simply no time to wait for laboratory
results, which can take up to 72 hours before the complete picture,
including a potential antibiotic-resistance profile are available [30–34].
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Epidemiology
In the United States, sepsis is rated as the 10th leading cause of
death [15,21,26]. A 22-year American study showed that the number of
sepsis patients increased from 82 to 240 per 100,000 in 1979 and 2000.
Even though the overall mortality rate had decreased from 28% to 18%,
the total number of deaths was three times higher due to an overall
increase of incidence [21]. Globally, sepsis is estimated to affect between
15 and 19 million people every year [15–17]. In spite of having a quite high
disease burden (e.g. incidence, mortality and cost), sepsis still not attract
as much public attention as do diseases such as breast cancer and AIDS
[16,35–38].
Figure1.3: The relationship between SIRS, sepsis and infection. Sepsis is a systemic
inflammatory response syndrome (SIRS) caused by an infection. An infection may have
several different origins: bacteremia, fungemia, parasitemia and viremia. There are several
medical scenarios other than sepsis in which SIRS can appear. This image is reproduced
with the permission of the copyright holder [25].
According to the American Center of Disease Control and
Prevention (CDC), the mortality rate per 100,000 populations in 2010
was 25.9 for breast cancer, 2.5 for AIDS and 41.4 for stroke [39–41]. If we
compare these numbers with the equivalent values for sepsis mortality,
one can easily see why sepsis is one of the ten leading cause of death in
the US and the 2nd leading cause of death in non-coronary ICU
[21,26,30,35,42–44]. Martin et al. estimated the overall death rate due to
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sepsis at 43.9 per 100,000 for the year 2000 [21], while Wang et al.
estimated a mortality rate of 65.5 in a study ranging between 1999 and
2005 [45]. In contrast, Melamed et al. estimated 50.5 deaths per 100,
000 for the same period (1999-2005). Daniels et al. made a similar
comparison between sepsis and diseases with high public awareness in
UK (Figure 1.4) [37]. The incidence of sepsis in the European Union was
estimated to be 90.4 deaths per 100, 000. In comparison the incidence of
breast cancer that was determined to be 58 per 100,000 [37]. The
occurrence of severe sepsis in Europe, on the other hand, seems to lie
between 50 and 100 cases per 100,000 individuals [35,36,46–51]. These
differences can be attributed in part to seasonal variations, variations in
the length of study, and variations in the diagnostic criteria [42].
Although it might be difficult in some cases to determine the true
sepsis incidence/mortality rates, these studies still confirm its place
among the common causes of deaths worldwide. Sepsis deserves
increased attention equal to AIDS, prostate cancer, breast cancer and
other better-known conditions.
Figure1.4: Mortality rate for various diseases in the UK This image is reproduced with the
permission of the copyright holder [37]
Misdiagnosis: a fatal error.
As mentioned earlier, the definition of sepsis is quite broad and to
some extent overlaps with other diseases. This definition was established
as recently as in 1992 by the ACCP/SCCM conference committee [25].
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But does it help or is there still a lot of confusion? One thing that
is absolutely certain and is agreed by all is that speed saves lives: the
sooner a potential sepsis patient is identified the better the survival
chances. So how is this translated into the clinics and hospitals around
the world? An interesting survey performed by Poeze et al. looked into the
perception, attitude and the ability of healthcare professionals around the
world to diagnose sepsis [52]. The great majority, 86% of the physicians
were of the opinion that sepsis could easily be misdiagnosed and 65%
believed physical examination to be inadequate. Only a small fraction of
the participants (22%) used the ACCP/SCCM criteria to define sepsis and
this eight years after the definition was set [52]. The problem with the
criteria is that they have high sensitivity but low specificity. A vast
majority of the patients in ICUs and general wards fulfilled these criteria
at some point [53]. A new attempt was made in 2001 by the ACCP/SCCM
conference to further improve the sepsis criteria [54]. But it does not
seem to have had the desired effect. A comparative study showed little
difference between the 1991 and the 2001 criteria [53]. Indeed, much
depends on the clinician’s ability to predict sepsis. Consequently, the
confusion around sepsis criteria is worrisome, since any delay in
treatment can severely reduce the chances of survival [30,55–60]. Kumar
et al. have shown that chances of surviving septic shock decreased by 8%
for every hour the correct treatment is delayed [58].
Current diagnostic assays
Although today’s microbiological gold standard, blood culturing,
is highly affected by external technical factors (proper skin preparation,
sample volume, transport time, incubation atmosphere, blood-to-broth
ratio, culture media and so on), no method has yet been able to replace it
fully [30,61–66]. The main challenge of microbiological diagnostics is the
low bacteria load in the sample. As the clinical signs become manifest, the
blood stream still contains as few as 1-30 colony-forming units (CFU) per
ml [33]. The bacteria load might increase to 1000 CFU/mL, but this is
encountered only in severely ill patients [67]. According to Towns et al.,
almost 50% of all patients have less than 1 CFU/mL [10]. Hence,
sufficient sample volume is a highly important parameter.
The ability of blood culturing to detect only viable bacteria can be
seen as both strength and a weakness. Blood contains antimicrobial
19
agents that inhibit bacterial growth. This inhibition is further increased in
patients with antibiotics already in their systems, which give rise to false
negative results [68]. Although the sensitivity of blood culture is
considered to be 1 CFU/mL, only a third to a half of all sampled septic
patients yield positive cultures [10,31,55,69–71]. The difference in yield is
directly associated with the factors cited above, but the major drawback is
the time it takes to grow enough cells needed for analysis (up to 72
hours). However, classic microbiological methods are far superior when
it comes to determine antibiotic-resistance profiles. Molecular methods
will give a yes/no answer based on already known resistance
mechanisms/genes. However, the absence of a resistance gene will not
necessarily mean the organism is susceptible to a particular antibiotic.
There is seldom a situation where a single gene can give rise to resistance,
since phenotypic resistance to a certain antibiotic may be caused by a
whole array of different genes. For example, the resistance to betalactams among the Enterobacteriaceae family has been attributed to
several hundred mechanisms [72]. False positive signals will occur for
silent genes or pseudo-genes since resistance is dependent not only on
the presence of the gene but also in its expression level. Furthermore,
genetic methods will not give any information regarding the minimal
inhibitory concentration (MIC). The occurrence of false negatives is also a
possibility as in the case of primer binding-site mutations [73–76]. There
is also the barrier of sample preparation for complex samples, which
might contain assay inhibitors. However, molecular methods open up the
possibility of circumventing time-consuming culturing thereby decreasing
the turnaround time. They may also be very advantageous when it comes
to slow-growing and fastidious organisms since they can hardly be
detected with today’s gold-standard method, thus giving rise to false
negative results. Even though the idea of a universal molecular-based
method for detecting all bacteria with all possible combinations of
antibiotic resistance may well be overly ambitious, developing a rapid test
for a few clinically relevant strains is not. Even rapid gram determination
would be useful in clinical settings, as it could narrow down the antibiotic
spectrum.
Nucleic acid-based techniques
A number of the nucleic-acid-based techniques (NAT) for sepsis
diagnosis are available on the market today. These methods can be
divided into two groups: techniques that involve cell enumeration
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(positive cell culture) and techniques that directly use blood as a sample.
Nucleic-acid-based techniques can be sub-divided in function of their
analysis: pathogen-specific assays, universal broad-range assays and
multiplex assays [30,33]. There are a number of parameters to take into
consideration when evaluating these tools: the actual hands-on
time/number of assay steps, assay performance when it comes to polymicrobial samples, the total turnaround time, sensitivity, and specificity.
Particularly in the context of sepsis, however the most important
parameter is the extent of its diagnostic spectrum. A common barrier,
regardless of the nucleic-acid-based approach, is sample preparation
(which will be discussed in more detail in the following chapter), and a
common limitation is the antibiotic-resistance profile. This is also the
reason why molecular methods are seen as a compliment to the gold
standard.
Positive blood culture
While all of the techniques discussed in this section require a preculturing step, they differ in their diagnostic spectrum, turnaround time
and actual hands-on time. As can be seen in Table 1.1, these assay
methods have their strengths but, as yet, none provide a complete all-inone-tool. One must compromise between the number of target species,
the assay time, and the number of resistance markers. Two of these
methods (Verigene and Filmarray) provide a closed-box system with
minimal hands on time (5 minutes), which is basically what one looks for
in a new diagnostic tool, but there is still room for improvement when it
comes to the diagnostic spectrum.
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Table 1: 1 Techniques that require positive blood culture.
Method
Analysis
Multiplex
Nr. of
pathogens
Resistance
genes
Handson time
Assay
time
Prove-it
sepsis
[7,9,61,77]
Multiplex
PCR,
microarray
colorimetric
read-out
Yes
74
3
90 min
3-3.5h
PCR based
No
24
3
5 min
1h
Microarray
Optical
detection
Yes
13 gram+
5 gram -
3
5 min
2-2.5h
Film Array
[9,78]
Verigene
[9,71,79]
Diagnosis directly from blood
It would be highly advantageous to eliminate the blood-culturing
step and identify the disease causing pathogen directly from blood.
Consequently, the turnaround time could be drastically reduced and
specific antibiotic therapy could be administered. The first attempt to
isolate bacteria from blood was made in 1993 [77]. There are a number of
assays available on the market that offers pathogen detection/isolation
directly from blood: Septifast (Roche), Septites (Molezym), MagicPlex
Sepsis Real-Time Test (Seegene), Vyoo (Sirs lab) and Polaris (Biocartis)
are a few examples. SeptiTest from Roche has been available on the
market since 2004 and detects 19 of the most common bacteria and six of
the most common fungi [78]. The newest addition is Polaris from
Biocartis, a platform that is currently under development. One of the
interesting aspects of Polaris is that it selectively removes human DNA
(deoxyribonucleic acid) before the pathogen lysis takes place. The
selective removal of contaminating human DNA or enrichment of
bacterial DNA prior analysis is a strategy used not only by Polaris
(Biocartis). Both Molzyme and VYOO (SIRS lab) remove human DNA or,
as in the case of VYOO, enrich bacterial DNA by using an affinity
chromatography (PureProve) [30,79,80]. PureProve (SIRS lab) uses
characteristic motifs of prokaryotic DNA, non-methylated CpG
(cytidylatephosphate-deoxyguanylate) motifs, to bind bacterial DNA in a
resin while human DNA is washed away [80]. In a study by Loonen et al.,
the Polaris chemical-based method is compared to Molzyms’ (MolYsis)
enzymatic removal of human DNA [81]. To my knowledge no other
reports have been published about the Polaris platform. An interesting
22
study that was recently published used the DNA-binding section of a
bacterial topoisomerase (Gyrase) to selectively isolate prokaryotic DNA
[82].
Table 1.2 summarizes the differences between these assays. Two
things are striking in this table: the first is that all of the methods use PCR
(polymerase chain reaction) to some extent; the second is that the size of
the sample volume (1-5mL) is very low compared to the gold standard
(20-30 mL) [33,83]. According to Jordana-Lluch et al., low volumes are a
necessity when working directly with blood because of the presence of
large amounts of human DNA, which may hinder detection or inhibit the
PCR reaction. Moreover, there are several natural components in blood
besides leucocyte DNA that might reduce the PCR capacity, one such
being hemoglobin [83–85]. It has also been shown that even the presence
of immunoglobulin G (igG) could have possible inhibitory effects [84–
86]. The list of inhibitors makes the sample preparation especially
important for it concerns not only isolating the pathogen but also to
getting rid of any inhibitory substances. Another component affecting the
assay outcome, apart from the various inhibitory substances, is the choice
of DNA polymerase (a crucial component in PCR). DNA-polymerases
differ in their capacity to withstand the presence of inhibitors in blood.
Al-Soud et al. found that AmpliTaq Gold was highly sensitive to the
presence of blood. An amount of 0.004% (vol/vol) blood resulted in
complete inhibition [84]. Other polymerases (HotTub, Pwo, rTfl and Tli )
could tolerate up to 20% (vol/vol) blood [84]. Human DNA is not the only
negative aspect when working directly with blood. Nucleic acid-based
tests (NAT) risk producing medically irrelevant findings due to the
presence of circulating bacterial DNA, transient bacteremia and dead
bacteria
Recently, Laakso and Mäki have shown that the Prove-It sepsis
technology from Mobidiag could be used to analyze 1 mL of spiked wholeblood samples. Together with two other technologies, the SelectNA bloodpathogen isolation kit (Molzyme) and the Nordiag Arrow automated
extraction device, they were able to detect a number of different bacterial
species with a detection range of 11-600 CFU/mL (depending on the
species) [87].
23
Table 1.2: Diagnostics directly from blood
Method
Volume
Analysis
LOD
CFU/mL
Nr. of
Pathogens
TAT
SeptiFast (Roche)
1.5mL
PCR, Melting
curve analysis
3-30
25
6h
1-5mL
PCR, Sequencing
>300
612
5mL
PCR, gelelectrophoresis
3-10
~40
68h
1mL
3x PCR reactions
required
N/A
27
6h
1-5mL
PCR, real time
fluorescence
N/A
N/A
N/A
SeptiTest
(Molzym)
[9,83]
VYOO/LOOXTER
(SIRS lab)
[7,9,77,81,92
MagicPlex
(Segeene)
[77,83,85,93]
Polaris (Biocartis)
[83,94]
N/A
24
CHAPTER 2
Isolation techniques for complex fluids
“Blood is a treasure of information about the functioning of the
whole body. Every minute, the entire blood volume is recirculated
throughout the body, delivering oxygen and nutrients to every cell and
transporting products from and toward all different tissues. At the same
time, cells of the immune system are transported quickly and efficiently
through blood, to and from every place in the body where they perform
specific immuno-surveillance functions. As a result, blood harbors a
massive amount of information about the functioning of all tissues and
organs in the body”.
- Mehmet Toner and Daniel Irima (from Blood on a Chip) [88].
25
The Challenge: Taking Blood Apart
Toner and Irima’s observation above describes the importance of
blood and the potential amount of information hidden within it.
However, accessing that information is challenging, the main one being
the complexity of blood itself relative to the low number of target
molecules (Table 2.1). One milliliter (mL) of blood contains 5 × 109 red
blood cells (RBC), 2-5 × 108 platelets, and 5 to 10 × 106 white blood cells
(WBC) [88], while clinically interesting samples often have very low
target concentrations. One such example, besides bacteria and bloodstream infection, are circulating tumor cells (CTC), which are of great
interest within cancer research, but the number of target cells could be as
few as 10 cells/mL blood [89]. Another example are basophils in
allergology, which are mast cells that comprise less than 1% of the total
number of white blood cells [90] These two examples have one thing in
common: the low number of target cells in a sea of different blood cells so
sample preparation plays a significant role in each application area.
Sample preparation has often been described as the “bottleneck” or the
“road-block” for new technologies as well as the “forgotten beginning”.
Table 2.1: Number of cells found in mL of blood.
Cell-type/Target molecule
Cells/mL
RBC
5 × 10
WBC
Ref
9
[88]
5 to10 × 10
6
8
Platelets
2-5 × 10
Basophil
3
10 -10
Circulating tumor cells (CTC)
0,1-10
Bacteria (Blood stream
infection)
1-1000 CFU /mL
5
[88]
[88]
[88]
[89]
The goal of sample preparation is to separate target cells from
their surroundings in order to remove inhibitory substances that may
hamper downstream analysis while reducing the heterogeneity, the
complexity, of the sample itself [91]. Microfluidics brings promises to re-
26
shape the current health-care system by transferring diagnostic tools
from central laboratories to the vicinity of patient. To achieve this, sample
preparation must be included in the workflow, which would be an
improvement over the current situation in microfluidics in which “ofchip” macro-scale solutions are often the only ones available [92]. The
ideal scenario would be a “plug-and-play” system, an objective that many
researchers are striving to achieve [31,93].
Microfluidics – A Laboratory Time Saver?
As the word implies, microfluidics is about handling and
manipulating fluids in micro-scale dimensions. Here, micro technology,
engineering, physics, chemistry and biotechnology overlap. Microfluidic
aims to replace tedious laboratory work that often requires repeated
pipetting by one single automated closed box or hand-held device. Other
important objectives are high-throughput and multiplexing. Imagine the
advantage of screening multiple targets and running numerous tests
simultaneously. Throughput is defined as the number of assays a system
can perform during a certain period of time [94]. In the macro-world, the
ability to run 96 or 384 samples at once is considered high-throughput.
Now, with the aid of microfluidics, throughput can be moved beyond the
micro-plate. High-throughput microfluidics can be achieved serially or in
parallel. With parallel processing, a high number of samples can be run
simultaneously in order either to reduce the overall processing time or to
increase multiplexing by running different assays simultaneously.
Serially, throughput is achieved by taking a sample from processing to
analysis, that is, different steps and functionalities are integrated serially
in one setup. This is usually the case in Point-of-Care systems (Chapter
3).
The economic benefits of miniaturization are obvious: smaller
size would inevitably mean less reagent consumption, less waste and less
manufacturing cost [95–98]. When moving into the sub-millimeter scale,
there are certain characteristics that become prominent, one such being
laminar flow. Although Figure 2.1 is not a micro-scale example, it gives
quite a striking image of how two stream lines in a laminar-flow condition
would look. In such a system, mixing is caused primarily by diffusion.
27
Figure 2.1: An example of how a laminar flow streamline would look like in a microfluidic
device. Two parallel streams flow without mixing. Photograph taken by Jozef Kowalewski.
When designing physical systems that involve fluids it is
important to be able to predict the behavior of the flow.
The
dimensionless Reynolds number (Re = ρvd/µ) describes whether or not a
flow can be considered turbulent or laminar. The Reynolds number is in
essence the ratio between inertial to viscous forces [99]. A high Reynolds
number (>4000) indicates turbulent flow, while a low Reynolds number
(<2300) gives laminar flow. The intermediate values (2300<Re<4000)
indicate flow with both the laminar and turbulent flow regimes present
[100]. In the laminar regime, the flow will have a parabolic profile. Our
blood vessels are one example where this occurs. Here, layers of the blood
cells travel parallel to the vessel wall in an orderly fashion (Figure 2.2)
with no disturbance between the streamlines [101]. In contrast, in
situations where turbulent regimes take place, there will be swirling
motions and more unpredictable mixing. High flow-rates will generally
result in turbulent flows [102].
28
Figure 2.2 Parabolic flow profile in a blood vessel. Blood travels parallel with no
disturbances between the streamlines (arrows). The maximum velocity (Vmax) occurs at the
center line, and the lowest velocity is found by the vessel wall (V=0).
Smaller reaction chambers give shorter diffusion paths, which in
turn enable more rapid reactions than their macro-scale counterparts can
provide [103]. One example of this is conventional enzyme-linked
immunosorbent assay (ELISA), which is typically performed in microtiter plates with mm-scale diffusion distances. As a consequence, an
ELISA can take from several hours up to 2 days to complete. Studies have
shown that a microfluidic setup can reduce the assay time from hours to
minutes [103–106]. A high surface-to-volume ratio is another scalingdown effect that could be advantageous, especially for surface-bound
affinity assays [107,108].
Low cost, fast reactions, high-surface-to-volume ratio,
multiplexing, high-throughput, and automation are keywords that are
usually positively associated with microfluidics. However, there are also
challenges when working in micro-scale dimensions (Table 2.1).
Depending on the objective, a laminar flow regime can be strength or a
weakness. On the positive side, it provides more precise placement of
particles and reagents, thus making multiplexed chemical dilutions
possible [97]. On the downside however, mixing becomes challenging as
the driving force is mainly diffusion-based and occurs at stream-line
interfaces [97,109]. The disadvantage of a high surface-to-volume ratio is
that there are more surfaces available for adsorption, which leads the
discussion to the choice of material. The material most often used in the
field of microfluidics is polydimethylsiloxane (PDMS). One of its
advantages is biocompatibility so it has been used in catheters, drainage
tubing, pacemakers, prostheses and various implants (blood vessels,
heart valves, breast implants) [110]. Biocompatibility is to a large degree
29
determined by cellular responses, which in turn are based on their
reaction to adsorbed proteins on biomaterial surfaces. When an implant
is made, the very first event is protein adsorption. This takes place within
seconds of implantation, thereby making the biomaterial a biologically
active surface. Therefore, cells in our bodies do not encounter the
biomaterial itself but the proteins adsorbed on its surface [110]. PDMS is
hydrophobic in its nature. To avoid non-specific adsorption, various
surface modifications are needed [104], which is particularly important
when working with blood [111]. Blood plasma encompasses a myriad of
proteins readily adsorbed on surfaces. Among these proteins there are
some (fibrinogen, fibrnoectin, vitronectin, von Willebrand factor, etc.)
that induce platelet adhesion. Surface modification may help evade
blood-cell activation, platelet adhesion, and coagulation upon bloodsurface contact [110,112]. Such modification may involve heparin or poly
(ethylene glycol) (PEG).
Table 2.2: Pros and cons of using microfluidics.
Pros
Multiplexing
High-surface to volume ratio
Automation
Faster reaction
Fluid control
Low cost
Cons
Mixing
Non-specific adsorption
Interfacing
Clogging
Bubbles
Microfluidic-Based Separation
Traditionally in microfluidics, separation has been divided into
active and passive forms. Simply put, an active separation requires an
external force while a passive separation relies more on channel geometry
and inherent forces [113–115]. Table 2.3 gives an overview of the different
active and passive separation methods as well as the separation criteria
used in them. These methods are beyond the scope of this thesis. For
further reading see the suggested references [113,115–118].
Different cell characteristics are often used to differentiate the
target from its surroundings: size, density, deformability, surface antigen,
30
surface charge and cell-wall composition. In the following section, the
focus will be on cell-wall composition and size.
Table 2.3: Active and passive microfluidic separation
Category
Method
Acoustophoresis
Dielectrophoresis
Active
separation
Electrophoresis
Optical
Hydrodynamic
Magnetic
Passive
Separation
Deterministic
lateral
displacement
Inertial
Filtration
Separation
criteria
size,
density,
compressibility
Surface
charge,
density, size
Surface charge
Size, refractive
index,
polarizability
Size
Magnetic
susceptibility
Size,
deformability
Ref
Size
Size
[113,116,120,121]
[113,116]
[113,116–119]
[113,117,118]
[113]
[113,118]
[113,115]
[116]
[113,114,116]
Cell-Wall Composition
Exploiting differences in cell-wall composition is, perhaps, one of
the first approaches that come to mind when separating targets from
their surroundings. Antibody-based systems would be one of the most
obvious implementations. Antibodies are proteins that are part of the
body’s defense system as well as being invaluable tools in today’s modern
diagnostic toolbox [122–125]. These proteins bind targets called antigens
with high specificity (“lock and key”) and sensitivity even in complex
solutions [105]. Assays that involve antibodies are called immunoassays.
There is a large selection of different types of immunoassays
(e.g.,enzyme-linked,fluorescent-based, chemiluminescent-based and
radio immunoassays). Perhaps the most important aspect of antibodies is
the ability to custom-make them against almost anything with high
affinity. Ever since they were introduced in the late 1950s, they have
31
become standardized tools, and there are a great variety of them [126].
You can find them coupled to fluorophores, enzymes, quantum dots, and
beads of different sizes and materials. It is, therefore, not surprising that
the annual sales of immunoassay material have been estimated to be as
high as $7.2 billion worldwide [123].
A less obvious tactic when using differences in cell-wall
composition is a chemical approach (selective lysis) that takes the whole
cell-wall into account as opposed to only certain surface molecules. This
method takes advantage of the large difference between the protective cell
barrier of bacteria and blood cells. First, blood cells lack a cell-wall. The
enclosing barrier of a blood cell is a cell membrane. A fundamental
function of the cell membrane is to segregate the liquid interior of the cell
from the watery environment outside the cell [127]. However, since
bacteria often live in harsh conditions, they need the extra protection
against the environment that comes in the form of a cell-wall [128]. For
instance, Escherichia coli can be found in the mammalian gut and
Salmonella in the gall bladder [128]. Here, the bacteria must be able to
tolerate detergents such as bile salts and gastric juices.
This difference in cell-wall/cell-membrane composition has been
used in both macro- and micro-setups. In addition to centrifugation as a
means of blood fractionation, there are also chemical methods that
selectively lyse red blood cells, a process that typically involves
ammonium chloride [88,129]. This process is called hemolysis and has
been used extensively in the study of white blood cells (WBC). Here, cell
separation/isolation is necessary since the WBC make up only less than
1% of the whole blood [115,130]. With a selective lysis method, the
difference in cell-wall composition is used to lyse the majority of
erythrocytes with minimal damage to the leukocytes. In addition to
ammonium chloride as a lysis buffer, there are also a few commercially
available solutions: FACSlyse solution (Becton Dickenson), Molysis
(Molzyme), Zap-oglobin and Coulter Q-Prep lysis solution (Beckman
Coulter) [129]. Despite being commonly used, long incubation times with
ammonium chloride have been shown to activate leukocytes, thereby
changing their membrane expression pattern. Cell activation and cellmembrane alteration due to the isolation technique is always an
undesirable effect when attempting to study any cell type [129,131]. On
the macro-scale, lysing small volumes of blood (1mL) takes approximately
32
5 minutes. This is longer than necessary for an actual lysis event but it is
nonetheless needed due to macro-scale diffusion limitations [129]. This is
a perfect scenario where the advantages of microfluidics would come in
handy.
As mentioned earlier, bacteria cells distinguish themselves from
mammalian cells by having a cell-wall (as opposed to only a cellmembrane). Traditionally, bacteria have been categorized as grampositive or Gram-negative on the basis of the Gram-stain. Those bacteria
strains that maintain the dye are called Gram-positive and those that do
not are defined as Gram-negative [128]. The rough and smooth serotype
is a gram-negative trait that is based on their lipopolysaccharide (LPS)
structure. LPS is found in abundance in the outer membrane (OM) of
gram-negative bacteria and can be divided into three regions: Lipid A,
core-oligosaccharide and polysaccharide (O antigen) regions. Bacteria
that lack the polysaccharide part (O antigen) of the LPS molecule are
called rough strains [69,128,132,133]. What follows is a schematic
overview (Figure 2.3) of the differences between the cellular barrier of
erythrocytes, leukocytes and Gram-negative bacteria, which is a
fundamental difference that is taken advantage of in these macro- and
micro-based approaches.
Figure 2. 3: Schematic overview of differences between the cellular barrier of red-blood
cells, white-blood cells and Gram-negative bacteria. The cell membrane of RBCs and
WBCs consists mostly of phospholipids while a Gram-negative cell-wall has an outer
membrane, a peptidoglycan layer and an inner membrane.
33
Chemical approach
Sethu et al. showed complete lysis of erythrocytes with
ammonium chloride after only 30 seconds in a microfluidic device. The
throughput of this particular device is quite low considering the small
flow-rate (3.5 µL/min) [129]. In a later publication, Sethu et al. used
deionized water to lyse erythrocytes selectively: they managed to lyse all
of the RBC while keeping the WBC in a near non-activated-state,
something that is considered difficult to achieve in a macro-scale setting
where longer incubation times will inevitably activate or lyse cells. All in
all, it takes 30 minutes to process 0.6 mL blood (20µL/min), which is
approximately a 6-fold improvement from the previous setting [131].
The strategy of selective cell lysis was further improved in one of
our studies. By taking advantage of the differences between the blood cell
membrane and bacteria cell-wall, we managed to lyse both erythrocytes
and leukocytes selectively while keeping the target of interest, the
bacteria, intact and viable. This will be further explored in the present
investigation section (Chapter 4).
Hwang et al. used another interesting approach in which they
treated bacteria samples with sodium acetate to induce adherence. To
increase the surface-to-volume ratio even further, a microfluidic device
containing pillar arrays was fabricated. A capturing efficiency of 70% was
reached for all the sodium acetate samples within the concentration range
of 103-107. Bacteria (107 CFU/mL) spiked in 50% blood (diluted with
sodium acetate) reached a capturing efficiency of 40% [134]. In a followup article, Hwang et al. reported a complete assay by executing everything
from sample preparation to PCR on the chip. Here they reached a
capturing efficiency of 40% for bacteria samples (104- 107 CFU/mL)
spiked in blood with a flow rate of 100-200µL/min [135].
Affinity-Based Approaches
There are a few interesting research articles that use affinitybased assays in a microfluidic system to isolate bacterial cells from blood
[136–138]. One of the most interesting approaches has been used by
Daniel Kohane’s laboratory at the Boston’s Children Hospital [136], a
synthetic ligand, zinc-coordinated bis(dicopolylamine) (bis-Zn-DPA) with
high affinity toward both gram-negative and Gram-positive bacterial cellwalls [136,139,140]. As a proof of concept, they were able to isolate E.coli
34
with a concentration of 106 CFU/mL from whole blood using a flow rate
of 60mL/h [136]. For this to be applicable in a diagnostic setting the limit
of detection must be improved and the binding properties of bis-Zn-DPA
with a range of different bacteria species, both Gram-positive and gramnegative, must be tested. However, they did use whole blood as a sample
and were able to process the blood quite rapidly (60mL/h) [136]. This
ligand was first described in 1964 [139] and has been extensively used by
Bradley D. Smith’s laboratory at University of Notre Dame (USA) [139–
143]. Here, they demonstrated both the binding of this ligand against
both Gram-negative (smooth and rough serotype) and Gram-positive
bacteria. Interestingly, they reported a change in the binding properties
of bis-ZN-DPA when conjugated to quantum dots. In this configuration,
the ligands were only able to bind rough-mutants of E.coli. They also
failed to bind any of the Gram-positive prototypes they used, which they
attributed to the overall size of the conjugated ligand (15-20 nm), a size
too large to enter the pores in the cell-wall (maximum 10nm in diameter)
[143]. In the work of Lee et al. (Kohane’s lab), E.coli Stbl3 was used,
which is a derivate of E. coli HB101 [136,144,145]. E.coli HB101 is K-12
derivate with a truncated form of LPS, a rough serotype [128,146]. As a
comparison, it would be interesting to know if their setup can be applied
to both rough and smooth strains [136,144].
A recently published technical report from nature medicine
describes a recombinant opsonin-based method to cleanse blood from
bacteria and toxins. This recombinant Mannose-binding-lectin holds
promise of binding a large panel of different bacteria, fungi, viruses and
toxins [147]. The authors provide a unique interpretation of the sepsis
dilemma. They do not provide means of identification but a new
treatment direction for sepsis patients. If the assay sensitivity is able to
match clinical relevant sepsis cases (1-1000 CFU/mL, for symptom
showing patients) this could revolutionize treatment strategies. In a
constant battle against ever increasing drug-resistant pathogens, this
could indeed become a future strategy. Today this method has efficiently
cleansed blood containing a bacteria concentration of 104 CFU/mL and a
toxin (LPS) level of 10µg/mL. Sepsis patients have an endotoxin level of
300-400pg/mL [148,149].
35
Size-Based Approaches
Difference in size is another well-used characteristic for sample
separation. Deterministic lateral displacement (DLD) and filter-based
methods are two examples of microfluidic systems that use differences in
cell diameters as a separation criterion. However, these methods are
associated with clogging, fouling and low flow rates [116]. Another
method that involves size as a separation criterion is inertial
microfluidics. The most desirable feature of this method is particle
focusing at high flow-rates. As a consequence, it is possible to process
large volumes sample, which is not as time consuming as with other
microfluidic-based systems.
Inertial Microfluidics
With inertial microfluidics, particles travel across streamlines
instead of keeping their original inlet position as is seen with laminar flow
profiles. Inertial focusing brings the possibility of directing particles of a
particular size to a precise equilibrium position (particle focusing) within
the flow. As a consequence, particles of different sizes can be sorted out at
different outlets. These particle focusing positions arise at high flow rates
due to two counteracting forces that act on the particles: shear gradient
lift forces and wall-induced lift forces. Smaller particles such as bacteria
(1-3 µm) are more difficult to focus since they undergo smaller forces
than do the larger blood cells (8-20µm) (Table 2.4) and so maintain a
more uniform distribution [150]. However, there are a few articles within
the field of inertial microfluidics that focus on separating bacteria from
larger surrounding blood cells [99,151,152].
Table 2.4 Different cell sizes.
Cell-type
Size
RBC
8 µm in diameter x 2 µm thick
WBC
5-20 µm in diameter
Platelets
1-3 µm in diameter
CTC
16-20 µm
E.coli
1-3 µm in length
36
CHAPTER 3
Point-of-Care: The Final Goal
”Point of care testing describes testing using handheld or
benchtop technology, where the result will be used in the screening for, or
the diagnosis and/or the management of, disease. It is an alternative to
using the services of a centralized facility such as a laboratory”.
Christopher P. Price (from Disease Management & Health Outcomes)
37
Point-of-Care: An Overview
Point-of-care (POC) and lab-on-a-chip (LOC) are two major
concepts in medical microfluidics where “cheap”, “fast” and “reliable” are
the ultimate goals. The terms of themselves are quite descriptive but still
deserve a short conceptual overview. Lab-on-a-chip integrates several
laboratory functions into one or a series of miniaturized compartments,
providing a potential black-box system, where the sample goes in and an
answer comes out. Point-of-care, on the other hand, refers to devices used
in close-proximity of the patient. It could be a hand-held device used by
medical staff, a home monitoring system used by the patients themselves,
or a small bench-top technology that would reduce the dependence on
large central laboratory testing sites [153]. The driving forces behind
point-of-care development are a number of benefits such as rapid
decision making, early therapy initiations, improved treatment
optimization and reduced hospital stays [154] .
Point-of-care instrumentation can be categorized in two different
subclasses based on their target groups: those developed for resourcelimited settings and those made for developed countries [155]. The
limiting factor will be significantly different depending on the target
group with substantially different requirements. Consequently, all of the
devices that are considered “point-of-care” are not suitable for all
settings. Some e of the requirements of resource-limited settings are
robustness, environmental considerations (temperature, humidity),
portability, minimal hands-on-time, no need of highly trained personnel,
and easily interpreted results [155,156]. Taking all this into account, it is
easy to see that the spectrum of point-of-care devices is very large,
ranging from more advanced benchtop devices to dip-stick assays [153].
Regardless of the end user, the point-of-care field would greatly benefit
from simplified sample preparation[31]. Two examples of automatic
systems that have been mentioned in point-of-care reviews are
GeneXpert (Cepheid) and BD Max (BD Diagnostics), both of which use
off-line sample pre-treatment [93,157,158]. However, these systems
would not be well suited in resource-limited settings since they are high
in energy consumption and cost [159].
Numerous studies have shown that the turnaround time (TAT)
can be drastically reduced by POC instruments with respect to
38
standardized laboratory tests by removing transport time, sample
preparation (centrifugation, separation), validation, and the need to
forward results [153]. Although there are several investigations showing
improved TATs for point-of-care devices over traditional laboratory
testing, the reports on the actual therapeutic TAT and/or the impact on
hospital stay vary [153].
Operational Steps within Point-of-Care
One of the first objectives of a point-of-care system is the
reduction of the sample volume from mL scale to a µL scale, the so-called
macro-to-micro interface, while retaining the target of interest
throughout the entire process. This is not a small task since most analytes
often occur in low concentrations. Next, the sample needs to be cleared of
potential inhibitors and other abundant cells that might interfere with the
downstream analysis. Lysis is usually followed by a target amplification
step and, finally, the signal read-out (Figure 3.1) [160].
A possible alternative to conventional PCR is isothermal
amplification, which uses one amplification temperature (30°-65°C
depending on the method) thereby reducing instrumentation complexity
[158,160–162]. The final operational step is detection, which can be
achieved either at the endpoint (after the reaction) or in real time (during
the reaction). For systems aiming for low cost, endpoint analysis is more
appropriate than is to real-time analysis.
Point-of-care instruments would not exist without the joint effort
of microfluidic systems (the ability to miniaturize) and progress in
software-development. A technical concern that needs to be taken into
consideration within all of the operational steps is the need for fluid
control. This involves valves, mixing, fluid-movement, external/internal
heaters, coolers, choice of material, surface treatments and so forth
[155,163,164].
39
Figure 3.1 The Operational steps needed for a point-of-care system: sample preparation,
signal amplification and signal-readout. Adapted from Hartman et al. (2013) [160].
Point-of-Care for Bacterial Identification
The classes of analytes within point-of-care system vary from
proteins, cells and nucleic acids (RNA, DNA) to small molecules (glucose,
blood gases, electrolytes) [155]. There are, however, two interesting
platforms available for bacterial identification (Verigene and Film Array)
that meet the requirements of the automated operational steps for pointof-care devices.
Verigene
Verigene is a bench-top platform developed by Nanosphere, Inc.,
a company founded in 1999. The platform cartridges offer detection of
Gram-positive (BC-GP) and Gram-negative bacteria (BC-GN), yeasts and
viruses. The platform processes positive blood cultures (blood-stream
infection), stool samples (gastrointestinal infection) as well as whole
blood (for genotyping of cardiac samples) [165]. The most impressive
feature is the minimal hands-on-time needed (< 5 minutes + 2.5 hours
run-time).
Nanosphere’s patented technology consists of a sample processor
(SP) and a microarray reader. An assay requires three disposable units:
an extraction tray (for nucleic-acid extraction), a utility tray (containing
enzymes needed for enzymatic digestion) and the test cartridge (for
hybridization). The user simply loads the sample with a pipette onto the
first of the disposable cartridges and all sample preparation (lysis, DNA
fragmentation and isolation) and hybridization is then performed
40
automatically. An automatic pipette transfers samples on the extraction
and utilization tray before finally being moved to the test cartridge for
hybridization. The fluid movement within the test cartridge is done by
microfluidic channels and pumps [166–168].
Figure 3.2. A picture of the Verigene test cartridge (reagent pack and slide) together with its
Verigene processor and reader instrument. This image is reproduced with the permission of
the copyright holder [169]
Film Array
Film array (BioFire Diagnostics) is a multiplex PCR with
integrated sample preparation, amplification and detection. It requires
minimal hands-on-time. A plastic pouch, which contains several units as
well as freeze-dried material, is provided (Figure 3.3). The sample to be
analyzed is transferred from a syringe to a pouch. The movement of
samples within the pouch is controlled by pneumatic pumps [32]. In the
first unit, lysis is performed through bead beating. All of the released
nucleic acids are bound and transferred by magnetic beads. The Target
RNA is first reversely transcribed into DNA in a single large-volume PCR
reaction. Next, the diluted samples are transferred into small wells. Each
well is designed to detect one specific target. The analysis is done by endpoint melting curve data [32,170,171]. A positive blood culture with a
concentration range of 107 to 108 is needed [171].
41
Figure 3.3. Diagram of the Film Array system. The Film Array pouch (containing all of the
required materials) is loaded into a loading block (1). A solution is added through the blue
inlet port to re-hydrate freeze-dried reagents stored in the pouch (2). Next, the sample is
added at the red inlet port (4). Upon finishing these two steps, the Film Array pouch is
transferred from the loading block to the Film Array instrument where the entire assay is
initiated (5). This image is reproduced with the permission of the copyright holder [170].
Concluding Remarks
Although Verigene and Film Array platforms have very attractive
plug-and-play solutions, there is still room for further improvement.
Foremost, a blood culture step, which may take up to 72 hours, is still
needed. Another drawback is that both platforms are only able to run one
sample at a time. The number of detectable species, 14 for Verigene and
24 for Filmarray, is yet another important aspect. Only the Verigene
platform includes resistance markers (three antibiotic resistant genes for
Gram-positive bacteria and six antibiotic resistant genes for Gramnegative bacteria. In conclusion, these platforms are not able to cover all
possible organisms or resistance mechanisms [172–174]. They do,
however, show good performance with respect to traditional blood
culturing methods and have a more rapid turn-around time for their
pathogen panels [174].
42
CHAPTER 4
Present investigation
43
Aim of the Thesis
This thesis focuses primarily on sample preparation, with an
emphasis on bacteria isolation. The work behind this thesis consists of
three different approaches: (i) immuno-based isolation, (ii) selective cell
lysis, (iii-iv) size-based separation. An additional study investigated the
activity of a recombinant, Shigella spp Apyrase, which is an important
sample preparation tool in bioluminescence assays.
Paper I
This study investigates the possibility of specific isolation of
Gram-negative bacteria. To achieve this, antibodies targeting the
conserved region of the lipopolysaccharide (LPS) have been used. The
challenge lies in epitope unmasking. To improve epitope accessibility,
sample heat treatment was developed. The results show significantly
improved capture efficiency over non-treated cells.
Paper II
The bacterial cell-wall has a more rigid structure than does the
mammalian cell membrane and should, therefore, withstand harsher
chemical treatment. This physiological difference has been used to
selectively lyse blood cells while keeping bacteria intact and viable for
downstream analysis.
Paper III
By using inertial microfluidics, size-dependent particle focusing
at high flow-rates has been achieved. Particles with a diameter of 10 µm
are positioned at precise streamlines within the curved channel. The
focused particles can then be collected at a specific outlet with a
separation efficiency of 90%. As a proof of principle, white blood cells
were separated from diluted whole blood with an efficiency of 78%.
44
Paper IV
Elasto-inertial focusing is used to separate bacteria from blood.
With the use of non-Newtonian fluids, the blood components are diverted
to center of the channels while smaller bacteria remain in the side
streams and can subsequently be separated.
Paper V
The activity of recombinant Shigella flexineri apyrase (rSFA) is
compared to commercially available Solanum tuberosum apyrase (STA).
In terms of sample preparation, apyrase is an invaluable “cleanup-tool”
for bioluminescence assays where contaminating ATP needs to be
removed prior to an assay run. Initial studies show that rSFA has a higher
activity than does STA in buffer and serum.
45
Paper I
Epitope Unmasking for Improved Immuno-magnetic Isolation of GramNegative Bacteria
On a single Gram-negative bacterium, there are approximately 2
x
lipopolysaccharide (LPS) molecules, thereby making it one of the
major components of the outer cell membrane [69,132]. The LPS
molecule consists of three distinct regions: Lipid A, the core
oligosaccharide and the O- polysaccharides (Figure 4.1). There are at least
160 different O-polysaccharides serotypes for E.coli alone [175]. Bacteria
that somehow have lost the o-polysaccharide chain are called rough
strains, while those with a full length LPS are called smooth strains
[133,176].
106
The Lipid A region of this molecule is highly conserved within all
gram-negative bacteria, which makes it an interesting target for further
investigation [69,132,133]. Although the benefits of targeting the highly
conserved and abundant Lipid A portion is clear, there is an accessibility
challenge. Access to the Lipid A moiety is limited because it is partly
embedded in the membrane and thus is hydrophobic and also because of
the steric hindrance caused by the outer region of the LPS molecule and
the capsular polysaccharide [69]. This has been demonstrated by several
studies, which show that the binding of anti-lipid A antibody is interfered
with by the smooth full length O- polysaccharides [175,177–179]. The
steric hindrance presented by the LPS molecules also affects the binding
of antibodies to other cell surface antigens such as outer membrane
proteins (OMP) [180–182]. Membrane alternating antibiotics such as
ceftazidim have been shown to have a positive outcome on the binding of
anti-lipid A antibodies with the smooth chemotype [177].
46
Figure 4.1: An illustration of a Gram-negative cell-wall. The outermost layer of the outer
membrane consists of lipopolysaccharides (LPS). The LPS molecule can be divided into the
O-antigen, the core (inner and outer) and the Lipid A region [132].
Summary
To improve antigen accessibility, a heat treatment with different
temperatures was tested. An indirect immunofluorescence method was
used to verify the treatment effect. The results clearly show improved
binding between the antigen and the antibody after heat treatment. For
all strains, significant antibody binding could be seen around 60°C
(Figure 4.2).
Next, an indirect immune magnetic approach was used to isolate
bacteria from PBS (Figure 4.3). Bacteria cells were incubated with antilipid-A antibodies (Step 2) after being treated with heat treatment (Step 1,
60°C, 10 minutes). This was followed by an incubation step with protein
G-coated magnetic beads (Step 3). The isolated bacteria were then
analyzed with PCR.
47
Figure 4.2. Epitope unmasking by heat. An indirect immunofluorescence assay was used to
study the effect of heat on epitope retrieval. Clear improvement in
antibody-binding can be seen when bacteria cells were heat-treated,
especially around 60°C and 70°C.
As shown in Figure 4.4 the capturing efficiency was significantly
improved for samples subjected to mild heat-treatment (+AB 60°C) with
respect to the untreated samples (+AB RT). The level of non-specific
binding (-AB) was negligible for both the heat-treated and the untreated
samples.
48
Figure 4.3: Schematic overview of the immune magnetic bacteria isolation. A
bacteriasuspension (1) is subjected to heat to improve epitope accessibility. Second (2),
anti-lipid A antibodies are added with excess antibodies being removed by means of
centrifugation. The bacteria-antibody complex (3) is incubated with magnetic particles
conjugated with secondary antibodies. The bacteria bound on the beads (4) are collected
and analyzed with PCR
Figure 4.4: QPCR results for immuno-magnetic isolation of E.coli BL21 (~107 cells). A
significant difference in bacterial isolation can be seen between the room-temperature (RT)
and the heat-treated samples (60°C). An approximate efficiency of 81% was achieved while
the
untreated
cells
have
a
capture
efficiency
of
around
10%.
49
Paper II
Microfluidic Bacteria Isolation from Whole Blood for Sepsis Diagnosis
In this study, the difference between bacterial cell-walls and
mammalian cell membranes has been used to lyse blood cells selectively
while keeping the bacteria intact. Thanks to the more rigid bacterial cell wall, bacteria remain not only intact but also viable. To lyse blood-cells, a
combination of saponin and osmotic shock was used. Detergents such as
saponin have traditionally been used to permeabilize cell membranes and
can be found in plants and marine organism [183,184]. In this study the
susceptibility to osmotic lysis is further increased by saponin. As a result,
the more resistant white blood cells can be lysed as well.
Summary
As can be seen in Figure 4.5, bacteria are still viable after
treatment with 0.05-1% saponin and osmotic shock. At higher saponin
concentrations, the percentage of viable bacteria drops to 80%.
For the on-chip experiment, a saponin concentration of 1% was
used because of the incomplete lysis seen with 0.5% saponin and osmotic
shock (Figure 4.6). The difference between macro and micro is likely due
to the speed of mixing: mixing by vortexing has a more rapid effect than
does the herringbone structure used in the microfluidic chip.
50
7
Figure 4.5: Bacteria (~ 10 CFU/mL) and blood cells were submitted to different saponin
concentrations in order to determine the appropriate cut-off point where the bloods cells are
destroyed while the bacteria remain viable. A concentration of 0.5-1% seems to achieve this
goal.
Blood spiked with bacteria is added to Inlet 1 (Figure 4.6) while
the lysis solution containing saponin is added through Inlet 2. After
sufficient time has been given for thorough mixing between the blood
sample and lysis buffer, water was added through Inlet 3, to facilitate an
osmotic shock event. The flow rates (µl/min) of blood sample, lysis buffer
and water had a ratio of 1:1:2.
When the macro- to micro-adaptation was accounted for, the
same result was achieved, selective lysing of the blood cells while keeping
the bacteria intact and viable (Figure 4.7).
51
Figure 4.6 Microfluidic chip design. (A) This system has two functional sections: saponin
treatment (red) and osmotic shock (green). In both sections, a herringbone structure is used
to enhance the mixing. (B) The series of images illustrates the mixing efficiency of water
and fluorescein. As can be seen, complete mixing is achieved after three turns. The first
part of the chip is kept long in order to allow saponin and blood sufficient time to mix. The
lysis is terminated off-chip by adding PBS.
52
Figure 4.7: Selective lysis of blood components on the chip. A higher concentration of
saponin (1%) was needed in order to lyse all of the blood components selectively. The
bacteria were still intact and viable after this treatment. The bacteria concentration in these
experiments was 10 CFU/mL.
7
53
Paper III
Dean Flow-Coupled Inertial Focusing in Curved Channels
Inertial microfluidics uses inherent flow characteristics in
microchannels to focus and separate particles by size. Dominant inertial
forces (wall- and shear-induced lift forces) cause particles to move across
streamlines and occupy equilibrium positions along the faces of
microchannel walls. In curved channel geometries, an additional
secondary flow (Dean Flow) acts on particles and affects the particle
equilibrium positions (Figure 4.9).
Figure 4.9: An overview of the different forces at play in a microchannel with a curved
geometry. A homogeneous mixture of particles focuses into one single streamline as it exits
the 180° turn.
Summary
In this study, we introduced the design of microfluidic U-shaped
channels with varied widths to analyze systematically the 10-µm particle
behavior through curved channels. The height (50 µm) and the length
(20 mm before the curvature) were kept constant. Microfluidic U-shapes
channels with the following height:width ratios were fabricated: 1:1; 1:2;
1:5; 1:10 and 1:20. Particle focusing was achieved for the aspect ratios of
54
1:1 to 1:10. The focusing position was found to be independent of the
radius of curvatures.
Here, favorable scaling down properties were combined with high
flow rates to achieve continuous focusing of 10-µm particles in a device
with the aspect ratio of 1:10. A filtration efficiency of 92% was achieved
with the processing speed of 4.25 mL/min (Figure 4.10). The device was
further tested with biological samples: white blood cells were separated
from diluted whole blood with an efficiency of 78% while retaining high
viability (Figure 4.11). The flow rate used in this experiment was 2.2
mL/min. At this flow-rate, 98% of the 10-µm beads were collected at the
second outlet (Figure 4.11).
Figure 4.10: A. high-throughput (4.25 mL/min) filtration of 10-µm particles with an efficiency
of 92%.
55
Figure 4.11: (A). High throughput filtration of 10-µm particles with an efficiency of 96%.
(B) White-blood-cell filtration with a filtration efficiency of 78%. The flow rate in both
cases was 2.2 mL/min.
56
Paper IV
Elasto-Inertial Microfluidics Towards Bacteria Seperation From Whole
Blood for Sepsis Diagnostics
In paper III, inertial lift forces were used to focus 10µm particles
at a single streamline, hence facilitating their separation and subsequent
collection. Because of the size dependent nature of inertial lift forces,
smaller particles such as bacteria, will not experience an inertial-induced
effect. It is therefore not surprising that particles of 2 µm size have a
uniform distribution within the whole channel. To overcome this, the use
of “elasto-inertial” microfluidics was explored (paper IV). Here, nonNewtonian fluids (such as a high-viscous polymer) and lift-induced forces
are combined. As a consequence particles are focused away from the
equilibrium position towards the centre-line of the channel cross-section.
In such systems, larger particles can “migrate” away from the sample
matrix and into the polymer solution. The smaller particles, on the other
hand, will remain in the streamline and be effectively separated. Figure
4.12 shows a schematic overview of an elasto-inertial device for bacteria
separation.
Figure 4-12. Schematic overview of elasto-inertial based bacteria separation. The
viscoelastic flow enables larger particles to migrate towards the centreline of the channel.
Blood spiked with bacteria is flown and mixed with a polymer solution (PEO). Initially, all
blood cells remain in the streamline of which they entered but will eventually (after a certain
channel length) start to migrate into the polymer solution. The migration is strictly based on
size. The blood-cells are thereby focused at the centreline and can be fractionated out
through the middle outlet while all bacteria remain in the stream closer to the walls and are
separated through the side outlets.
57
Summary
The focusing behaviour of different particle sizes (10 and 2 µm)
was evaluated together with various polymer concentrations, channel
geometries and flow-rates. Based on experimental results micro-channel
dimensions of 50 µm in width, 65 µm height and a minimum of 25 mm in
length were found to be optimal. Figure 4.13 shows a successful focusing
of 2 and 10 µm particles by the time they exit the microchip.
Figure 4.13: Elasto-inertial focusing and sub-sequent separation of 2 and 10 µm particles.
As the particles reach the outlet, larger particles (green) have migrated away from the sidestream where they entered while smaller particles remain. Flow-rate (bead solution):
0.5µL/min. The flow-rate (non-Newtonian solution): 8µL/min.
Initial experiments shows promising separation results of
bacteria spiked in whole blood (Figure 4.14). At an optimum flow-rate of
0.25 µL/min, blood-cells are focused away from the side-stream toward
the centre-line. Bacteria are then collected both through the side and
middle outlet and plated on agar plates. As expected, the majority of the
bacteria cells are present at the side-outlets.
58
Figure 4.14: Plating results of bacteria spiked in whole blood and flown through
the channel. At the optimum flow rate (0.25 µl(min) , the bacteria are mainly
recovered through the side outlets. The data are from three independent
experiments.
59
Paper V
Recombinant Shigella Flexneri Apyrase Enzyme for BioluminescenceBased Diagnostic Applications
Bioluminescence is an attractive analytical method in many
biotechnological applications where light is emitted due to the actions of a
luciferase enzyme and
its substrate luciferin. Here, adenosine
triphosphate (ATP) drives the luciferase-mediated conversion of luciferin
to oxyluciferin, which generates visible light (Figure 4.12). Since cellular
ATP of a bacteria cell is relatively constant (one attomole), the luciferaseluciferin reaction can be used to determine the number of bacteria cells in
a sample. A vital step in such an assay is to eliminate any pre-existing
ATP in the sample matrix. To this end, apyrase is added prior to a
luciferin-luciferase reaction. Apyrase has the ability to sequentially
hydrolyse ATP to diphosphates (ADP) and monophosphates (AMP). The
most commonly used Apyrase is obtained from Solanum tuberosum
(STA), which is also called potato apyrase.
Figure 4.12: Luciferin-Luciferase
monophosphate and light.
reaction
yielding
Oxyluciferin,
The results show that RSFA depletes ATP at much higher rates
than does the potato apyrase (STA). Another interesting observation is
the level of ATP, which almost reaches zero for RFSA after only 10
minutes (Figure 4.13). This level of ATP depletion is never reached by
STA, which means that residual ATP from the sample matrix would
always be present in the subsequent reaction. The activity of these
60
enzymes was further tested in the presence of 10% (vol/vol) serum. A
simillar ATP depletion and activity rate was noted in serum (Figure 4.14).
Figure 4.13: An activity comparison between Solanum tuberosum (STA)
and Shigella flexneri (RSFA) apyrase in buffer. RFSA depletes adenosine
triphosphate (ATP) at a higher rate and to lower levels than does STA.
61
Figure 4.14: An activity comparison between Solanum tuberosum (STA)
and Shigella flexneri (RSFA) apyrase in 10% (vol/vol) serum.
62
Conclusion and Future Work
Paper I: The virulence of gram-negative bacteria has been
attributed to the Lipid A Region of the LPS molecule, which is also
referred to as an endotoxin. In the 1990s, attempts were made to produce
an anti-Lipid A antibody to neutralize endotoxins in patients suffering
from sepsis [133,175,185–187]. The basis of this strategy was to produce
cross-reactive antibodies, by taking advantage of the structure similarity
of the Lipid A region between different strains. However, the results were
contradictory [175,176,185,187,188].
In Paper I, the conserved region of the LPS molecule (Lipid A)
was explored. To increase epitope access, an epitope retrieval method
(without lysing the cells) was developed. Heat-treatment was shown to
have a positive outcome on the antigen access for the proof-of-principle
strains used in the study.
There are several possible ways to extend the work of Paper I.
First, it would be interesting to study the effect on pathogenic strains and
compare immune-magnetic isolation between treated and untreated cells.
Second, the treatment can be improved. Lipopolysaccharides are a tightly
packed forest on the outer layer of the cell-wall, covering 75% of its
surface [128,176,189]. This compressed state is made possible by
stabilizing cations (Ca2+, Mg2+) which neutralizes the repulsive forces
emitted by each LPS molecule [128,176,189,190]. With the use of
chelators such as ethylenediaminetetraacetic acid (EDTA), membrane
instability can be induced. An EDTA-induced release of the LPS
molecules without leading to a lysis event has been reported [190,191].
Whether or not such disturbance in the tightly-packed LPS forest leads to
improved antibody binding or not can only be determined
experimentally. Another interesting approach would be to use acetic acid,
which has been extensively used in protocols for counting white-blood
cells. Here, whole blood is diluted in an acetic acid solution to lyse red
blood cells prior counting the white blood cells. In hematology, this
solution is referred to as “Turks solution” [192]. An acetic-acid treatment
could offer a dual action: lysis of unwanted blood cells (making it easier to
work with complex solutions) as well as increasing epitope accessibility.
Mild acid treatment is known to cleave the linkage between Lipid A and
63
the core LPS [193]. This linkage exists in all LPS molecules and is called
KDO, 3-deoxy-D-manno-oct-2-ulosonic acid [193]. Bellstedt et al. have
used this process to create what they call “naked bacteria”, thereby
creating an immune-carrier for immunization [194].
Paper II: In Paper II, complete lysis of whole blood was
achieved while keeping the bacteria intact and viable. The presence of a
rigid bacterial cell-wall protects the bacteria from a lysis event [128].
Bacteria had full survival (100%) in the microfluidic chip for
concentrations from 107 to 106 CFU/mL. For concentration from 105 to
104 CFU/mL, bacteria showed a slight decrease in survival rate.
The work of Paper II can be further extended by including
parallelization in future versions of the device. Thus, samples can be
processed more rapidly. The retained bacteria viability is an interesting
trait worth further research. One suggested application would be to
combine this device with rapid antibiotic susceptibility testing [195,196].
Paper III: One of the challenges of microfluidic-based sample
preparation is the need to process large sample volumes. Often, a
compromise is made between speed and performance. In Paper III,
inertial microfluidics was used to focus and separate particles
continuously at high flow rates. Here, we used the synergetic effect of
inertial lift forces and Dean forces to focus particles based on size. An
efficiency of 96% was achieved for 10µm beads while the efficiency of
white blood cells was 78%. The flow rates used for both cases were 2.2
mL/min. Although inertial microfluidics show great potential for high
throughput blood processing, an inherent limitation for sepsis diagnostics
is the small bacteria size (1-3 µm), which makes them difficult to focus in
the presence of larger blood-cells.
To overcome this, two directions are envisioned: (i) the use of
“elasto-inertial” microfluidics (Paper IV) or (ii) integration with selective
blood cell lysis (Paper II).
64
Paper IV: By combining inertial-lift forces together with nonNewtonian fluids, larger blood-cells can be focused away from entering
side-streams toward the center of the microchip. As a result, blood-cells
can efficiently be removed from the sample. Since there is little or no
impact on the small-sized bacteria they will remain at the sideline.
Although a promising result, setting bacteria and blood cells apart,
further validation of the performance and separation efficiency is needed.
One struggling point is system clogging, making quantification through
plating difficult. In order to meet the need of high-throughput blood
processing, multi-parallel-channels can be fabricated.
Paper V: The efficiency of removing ATP traces is much higher
in Shigella flexineri apyrase (rSFA) than the commercially available
Solanum tuberosum apyrase (STA). Consequently, rSFA has the potential
of improving sample-preparation steps for bioluminescence assays and
thereby refining the detection limit even further.
65
Acknowledgement
I’ve learned a lot, been to a lot of places and more importantly
met a lot of wonderful, inspiring people. This has been the essence of my
Ph.D. journey and there are a lot of people I would like to show my
appreciation to. Thank you all for making these years memorable!!
Aman! First of all I would like to thank you for accepting me as
your Ph.D. student. Without you none of this would have been possible.
One has to search very long and very hard to find a more enthusiastic and
driven person. Thank you for all the interesting inspiring discussions. I
hope you reach your goal of providing valuable tools for resource-limited
areas.
Cell physics was my starting point and I have a lot of happy
memories from this division. A big thank you to Padideh and Athanasia
for making this environment extra quirky and full of energy. Padideh,
you’ve become like a sister to me. You’re the sweetest, most generous
person I know and I am truly happy that our roads crossed. A lot of
people have contributed in making cell physics feel like home; Ida, our
lovely downstairs (BioX) neighbor, thank you for patiently answering all
my physics related questions and dragging me to Zumba sessions (I
needed it!), Bruno, for always believing in me (!), Karolin for being
cheerful at all times (I especially liked our adventures in China), Elin for
all the fun we had together, Per for being your talkative self, Thomas F for
all the gossip and all the fika1 and last but definitely (!) not least Kate for
all the laughs we shared! You’ve become one of my closest friends and
partner in crime. I will never forget our attempts in making home-made
Christmas presents! It would have been a lonely lab without all of you. I
would also like to thank the PI’s: Hjalmar, Aman, and Björn for being the
foundation of this environment.
*Whoever visits Sweden for an extended time period will find it impossible to
avoid this activity/word: 1Fika, roughly means "to drink coffee/tea/," usually
accompanied by something sweet. Please note that it can be used as both a verb
and a noun.
66
Special thanks to all the members in the Intopsens consortium.
Even though my first years were filled with deliverables, tight timeschedules and a high pace, you all made it worth it. I enjoyed all our
meetings and discussions, especially Valencia 2010.
My Ph.D journey went from applied physics to the school of
Biotechnology. Although, I didn’t have time to properly get to know all of
you, I feel that biotechnology have become my home. Helen. Thank you
for creating such a fantastic environment. It’s always inspiring to see that
research and business can be combined!
I would like to thank all the former and present members of the
Nano-biotechnology group. You couldn’t wish for better colleagues and
friends. My best moments have been shared with many of you. I hope for
countless re-union events in the future. Special thanks to Emilie, Mary,
Lovisa, Jånas, Philippa, Eva (honorary member), Gustav, Prem, Staffan,
Cesc, Petter, Jorge and Dave for brightening my days. I would also like to
express my deepest appreciation to all of you (Sergey, Pavan, Jånas,
Asim, Mary, Zenib and Nilay) for helping me in my projects. This work
would not have been possible without your valuable input and
contributions. Sincere thanks goes to Andrés Veide, for graciously
handling my giggling attacks and for teaching me about bacteria work.
During my time at the school of Biotechnology, I especially
enjoyed my Albanova office, or more precisely the combination of people
in it (Emilie, Camilla, Daniel, Tarek, Thiru and occasionally Jesper). I
think we discussed everything between heaven and earth. Nothing was
too stupid. This office had quite a high threshold for everything, even
grumpiness and sarcastic remarks (always given with love) were quite
tolerated. I miss it still. Daniel, thank you for enduring my tendencies to
(slowly but ever so steady) take over your desk (I know it was hard on
you). Tarek. I very much like your fearless attitude in life, especially when
it leads to buying a sailing boat (with limited experience) and quickly
learning to master the art. A big thank you, to both you and Eva for letting
me join in on those adventures, it was great fun! Emilie, you have been
an invaluable friend and I admire your positive outlook on life. I
appreciate all your everyday gestures of kindness. Priceless!
67
Beyond the realm of Science (which sometimes seems like a
distant dream) I would like to tell all my Uppsala friends how much you
all mean to me. I will never forget Gotland and our wonderful ski trips. I
hope there will be many to come now that I will have somewhat more
time on my hands.
A big thank you to Edward, who knew that authors do strange
things but read my thesis anyway. Your emails have been a great source of
inspiration and your input and suggestion priceless.
Last but not least I would like to thank my family, my mom, dad, and my
sister for their unconditional love and support. I would not be the person
that I am, if not for you. You all mean the world to me!
To my extended family, thank you for taking care of me, for all
the relaxing moments, delicious home-made dinners and our many board
game evenings.
Above all I would like to thank Saeid, for making me laugh
whenever I needed it and for all your love and never-ending kindness.
There is no-one nicer and more understanding than you. I know, at times,
that my temper was particularly trying. You kept me sane during all the
ups-and downs of this journey. Love you so very much.
Some final words to all the lovely girls at Biotech (plan 3): You
rock!! Hope to see all of you on the dance floor!!!
68
Abbreviation
WHO
World health organisation
ICU
Intensive care unit
SIRS
Systemic inflammatory response
CDC
Center of Disease Control
CFU
Colony forming units
MIC
Minimal inhibitory concentration
NAT
Nucleic acid based techniques
DNA
Deoxyribonucleic acid
CpG
Cytidylatephosphate-deoxyguanylate
PCR
Polymerase Chain Reaction
IgG
immunoglobulin G
RBC
Red blood cells
WBC
White blood cells
CTC
Circulating tumor cells
ELISA
Enzyme-linked immunosorbent assay
PDMS
Polydimethylsiloxane
E.coli
Escherichia coli
LPS
Lipopolysaccharide
OM
Outer membrane
bis-Zn-DPA
Zinc-coordinated bis(dicopolylamine)
69
DLD
Deterministic lateral displacement
POC
Point-of-care
LOC
Lab-on-a chip
TAT
Turnaround time
LAMP
Loop-mediated-isothermal amplification
RNA
Ribonucleic acid
OMP
Outer membrane proteins
PBS
Phosphate-Buffered Salin
AB
Antibody
EDTA
Ethylenediaminetetraacetic acid
KDO
3-deoxy-D-manno-oct-2-ulosonic acid
70
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