1. No category

Sample Handling in Nanoscale Chemistry
Erik Litborn
Department of Chemistry
Division of Analytical Chemistry
Royal Institute of Technology
SE-100 44 Stockholm
Sweden
Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till
granskning för avläggande av teknologie doktorsexamen, fredagen den 26 maj, kl 10.00 i K2,
Teknikringen 28, KTH, Stockholm. Avhandlingen försvaras på engelska.
i
Sample Handling in Nanoscale Chemistry
PhD Thesis
© Erik Litborn, 2000
ISBN 91-7170-541-4
Royal Institute of Technology
Department of Chemistry
Division of Analytical Chemistry
SE-100 44 Stockholm
Sweden
ii
“The year is divided in
three periods:
before, after and during
the moose-hunt!”
iii
iv
Abstract
Sample Handling in Nanoscale Chemistry
Erik Litborn, Royal Institute of Technology, Department of Chemistry, Division of
Analytical Chemistry, SE-100 44 Stockholm, Sweden
Miniaturization is a strong on-going trend within analytical chemistry. This has
led to an increased demand for new technologies allowing smaller volumes of samples
as well as reagents to be utilized. This thesis deals with the use of open chip-based
reactors (vials); a concept that offers an increased flexibility compared to the use of
closed reactors. The vials are manufactured by anisotropic etching of silicon.
First, a short introduction is given on the benefits of performing chemistry in
miniaturized formats. Different types of reactors useful for performing chemistry in
nanoscale are described and the advantages and disadvantages of using a closed
contra an open system are discussed.
Precise dosing of nanoliter-sized volumes of liquids in contact mode is
performed by using miniaturized pipette tips or capillaries (Paper I, III&IV). Also,
non-contact dosing using piezo-electric dispensers is demonstrated by performing
nanoliter sized acid-base titrations (Paper II). Standard deviations on the order of 1%
were obtained.
Several strategies for handling the evaporation of water, while performing
tryptic digests of native myoglobin in low nanoliter sized vials, are demonstrated. An
increased conversion rate of the protein to peptides was observed when a nanovial (15
nL) reactor was used compared to the use of a conventional plastic vial (100 µL).
Principles based on reducing the driving force for evaporation (Paper III), continuous
compensation of evaporated material (Paper IV) as well as covering the reaction
liquid with a volatile liquid lid of solvent (Paper V) are used. The volatile liquid lid is
also used when performing PCR in volumes as low as 50 nL (Paper VI).
Short descriptions of the analytical methods utilized in the thesis; capillary
electrophoresis (Paper III, IV, V&VI), matrix assisted laser desorption/ionization
time of flight mass spectrometry (Paper I) and fluorescence measurements (Paper
II&VI) are presented.
Finally, an outlook of the developed technologies is given together with a
discussion concerning possible future requirements in miniaturized chemistry.
Keywords: capillary electrophoresis, continuous compensation, enzymatic
degradation, evaporation, lab-on-a-chip, liquid lid, MALDI, miniaturization,
myoglobin, nanoliter, nanoscale chemistry, parallel, picoliter, piezo-dispenser,
polymerase chain reaction, reactor, titration, tryptic digest, vial
© Erik Litborn, 2000
ISBN 91-7170-541-4
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List of Publications
This thesis is based on the following publications, which are referred to in the text by
their Roman numerals.
I.
S. Jespersen, WMA. Niessen, UR. Tjaden, J. van der Greef, E. Litborn, U.
Lindberg and J. Roeraade Attomole Detection of Proteins by Matrix-assisted
Laser Desorption/Ionization Mass Spectrometry with the Use of Picoliter Vials
Rapid Commun. Mass Spectrom. 1994, 8, 581-584
II.
E. Litborn, M. Stjernström and J. Roeraade Nanoliter Titration Based on
Piezoelectric Drop-on-demand Technology and Laser-induced Fluorescence
Detection Anal. Chem. 1998, 70, 4847-4852
III.
E. Litborn, Å. Emmer and J. Roeraade Chip-Based Nanovials for Tryptic
Digest and Capillary Electrophoresis Anal. Chim. Acta 1999, 401, 11-19
IV.
E. Litborn, Å. Emmer and J. Roeraade Parallel Reactions in Open ChipBased Nano-Vials with Continuous Compensation for Solvent Evaporation
Electrophoresis 2000, 21, 91-99
V.
E. Litborn and J. Roeraade A Liquid Lid for Biochemical Reactions in ChipBased Nanovials J. Chromatogr. B 2000, in press
VI.
E. Litborn, J. Rytte and J. Roeraade Miniaturization of the Polymerase
Chain Reaction Using a Liquid Lid of Octane BioTechniques 2000, submitted
Reprints are published with the kind permission of the journals concerned.
vii
viii
Table of Contents
1. General introduction concerning miniaturization
1
2. Reactors
3
2.1 Reactors based on closed systems......................................................................... 3
2.1.1 Capillary and column based reactors ............................................................. 3
2.1.2 Chip based reactors ......................................................................................... 3
2.2 Open systems.......................................................................................................... 5
2.2.1 Surface based reactors..................................................................................... 5
2.2.2 Chip based reactors ......................................................................................... 5
3. Dosing of liquids into open vials
7
3.1 Contact dosing........................................................................................................ 7
3.2 Non-contact dosing ................................................................................................ 8
3.3 Piezo-electric nanoliter titration (Paper II)............................................................. 9
4. Reactions in open chip-based vials
13
4.1 Nanorobot ............................................................................................................. 14
4.2 Methods for control of evaporation of water ...................................................... 15
4.2.1 Use of mechanical lids................................................................................... 15
4.2.2 Addition of non-volatile matrices ................................................................. 15
4.2.3 Increase of humidity (Paper III).................................................................... 16
4.2.4 Continuous compensation (Paper IV).......................................................... 17
4.2.4.1 Filling procedure
19
4.2.4.2 Evaporation theory for continuous compensation
20
4.2.4.3 Evaporation measurements during continuous compensation
22
4.2.5 Use of liquid lid (Paper V&VI) ..................................................................... 23
4.3 Influence of increased surface/volume ratio ....................................................... 28
5. Analysis of nL-pL sized volumes of sample
31
5.1 Capillary electrophoresis ...................................................................................... 31
5.1.1 Principle.......................................................................................................... 31
5.1.2 EOF ................................................................................................................ 32
5.1.3 Wall adsorption.............................................................................................. 33
5.2 Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry
33
5.2.1 Principle.......................................................................................................... 33
5.2.2 Sample preparation (Paper I) ........................................................................ 34
5.3 Fluorescence measurements ................................................................................ 35
6. Future outlooks
37
7. Acknowledgements
41
8. References
43
Appendix:
Paper I - VI
ix
List of Symbols
A
D
d
ddrop
d(κ)
E
h
hD
κ1
κ2
L
µe
η
q
r
Sh
v
W
area
diffusivity or diffusion coefficient
diameter
droplet diameter
difference of humidity
electric field
height
mass transfer coefficient
vapor pressure over a surface
vapor pressure in bulk air
length
electrophoretic mobility
viscosity of the separation media
ion charge
radius
Sherwood number
ion velocity
rate of evaporation
List of Abbreviations
ABS
C
CCD
CD
CE
CEC
CGE
CIEF
CITP
CZE
DNA
DOD
EOF
ESD
G
HPLC
LIF
MALDI
MECC
acrylnitrile buthadiene styrene
α-chymotrypsin
charged coupled device
compact disc
capillary electrophoresis
capillary electrochromatography
capillary gel electrophoresis
capillary isoelectric focusing
capillary isotachophoresis
capillary zone electrophoresis
deoxyribonucleic acid
drop-on-demand
electroosmotic flow
electrospray deposition
endoproteinase Glu-C
high performance liquid chromatography
laser induced fluorescence
matrix assisted laser desorption/ionization
micellar electrokinetic capillary chromatography
x
M1
M2
MS
myo
µTAS
PCR
PDMS
pI
PMT
SVR
T
theo
TOF
method for measuring the evaporation rate based on measuring
the time for one vial volume of solvent to evaporate
method for measuring the evaporation rate based on measuring
the flow rate in a solvent feeding capillary
mass spectrometry
myoglobin
micro total analysis systems
polymerase chain reaction
poly(dimethylsiloxane)
isoelectric point
photon multiplier tube
surface area/volume ratio
trypsin
theoretical values
time of flight
xi
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Sample Handling in Nanoscale Chemistry
1. General introduction concerning miniaturization
During the last few years, the need for new technologies to perform miniaturized
chemistry has increased dramatically. Typical applications are in areas such as high
throughput screening of drug candidates [1-3], combinatorial chemistry [4-6], genome
analysis [7], clinical chemistry and drug development [8] and proteomics [9]. For
performing a massive number of parallel reactions, it is obvious that miniaturization is
necessary and this requires adequate techniques for handling very small sample
volumes. Furthermore, in other areas such as material science, there is a growing
interest1 in combinatorial approaches, since a large number of experiments can be
carried out in a relatively small-sized scale [10, 11].
While the merits of miniaturized systems have been pointed out extensively [12, 13],
perhaps one particular advantage needs to be highlighted. In situations where chemical
reactions are to be performed, and the amount of material is limited, volume reduction
is very advantageous since high sample concentrations can be maintained in this way.
Under limited sample conditions, a dilution step will facilitate the sample handling but
consequently, it will result in slow reaction kinetics since the latter is dependent on the
concentration of sample and reagents. Furthermore, an excessive dilution will result in
poor detection sensitivity when the reaction is monitored with a concentration
dependent detector. Hence, the development of miniaturized systems, utilizing
technologies to handle small volumes of fluids are of considerable interest.
An additional aspect of miniaturization is the reduced cost for reagents. Chemistry
performed in small volumes allows the use of exclusive and very expensive reagents at
a reasonable cost. For example, an important procedure for protein identification and
characterization is to perform protein digests. The cost for performing one digestion
reaction, with trypsin in conventional volumes (50 µL) using protocols recommended
by the supplier (Sigma-Aldrich), is ca 30 SEK. A volume reduction down to 15 nL will
reduce the cost for the enzyme dramatically to ca 0.01 SEK.
Moreover, the increased surface area/volume ratio (SVR) associated with e.g. volume
reduction can provide an extra benefit if the solid surface is engaged in the reaction, as
a ligand carrier or as a catalyst. It is also preferable to enlarge the surface even further
when for example working with immobilized enzymes [14].
In addition, the development of miniaturized techniques has found an interest within
the area of industrial production of bulk chemicals [15] and impressive results have
been presented at the IMRET conferences2. However, such techniques will not be
discussed within the frame of this thesis since the devices utilized are designed for
much larger volume throughput than in the techniques presented here.
1
COMBI 2000, 2nd Annual Combinatorial Approaches for New Materials Discovery, January
23-25, 2000, San Diego, CA, USA
2
http://www.imm-mainz.de/content.html
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Erik Litborn
In pioneering work, the manufacturing procedures for miniaturized reactors were
limited to etching procedures in mono-crystalline silicon [16]. The etching procedures
for producing three-dimensional structures (micromachining) were developed from
the methods used within the electronic industry that manufactured integrated circuits.
Since then, micromachining has been performed in a large number of materials [17].
Micromachining has also been used for the production of molding masters [18], which
widens the range of materials used to include most polymer materials [19, 20].
Additionally, highly specific tools for handling solid materials e.g. nano-tweezers
based on carbon nanotubes [21], optical trapping [22] and laser catapulting [23]; have
been suggested.
In this thesis, the discussion will be focused on devices with applications within the
area of nanoscale chemistry (nanochemistry) which herein is defined as technologies
for handling and performing chemistry, using nanoliter to femtoliter sized volumes of
reagents.
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Sample Handling in Nanoscale Chemistry
2. Reactors
Chemistry, employing nanoliter volumes of samples and reagents can be performed
by using several different principles. It seems natural to classify the reactors in open or
closed systems when different unit operations are discussed. Alternatively, the
reactors can also be divided in reactions performed in continuous flow systems or
reactions performed in confined batch volumes [24].
In this thesis, the accessibility to a system and its reaction mixture, before (dosing of
reagents), during (“incubation”) and after (analysis) the reaction, is a most important
theme. In the present chapter, a short presentation is given of different closed systems
e.g. capillaries or etched chip-based channels as well as different open systems.
Advantages as well as disadvantages regarding the use of different reactor types are
discussed. This thesis presents work primarily carried out with open chip-based vials
as reactors since this concept fulfills important requirements of flexibility.
2.1 Reactors based on closed systems
2.1.1 Capillary and column based reactors
Capillaries are suitable to use as reactors, an inherent advantage since the reactor
volume is easily determined. Further, capillary-reactors can readily be made in
different materials e.g. glass, plastic, metal, and a wide range of diameters is available.
Enzymatic reactions have been successfully performed in capillaries with the enzyme
in free solution [25-29], with the enzyme immobilized on the capillary wall [30-35] and
with the enzyme immobilized onto a support medium [36, 37]. Capillary reactors have
also been used for PCR reactions in free solution [38-40] or with immobilized
templates in a capillary reactor [41]. Recently, Manz et al. presented a system where a
flow channel, made by etching a glass chip, was used for flow PCR [42].
A drawback of the capillary system is a limited flexibility in terms of accessibility,
since these are closed systems. Furthermore, even although a reaction can be carried
out in nL - pL sized volumes, sample transfer and handling procedures usually require
µL sized volumes of solution.
2.1.2 Chip based reactors
The use of systems based on microchips is an obvious approach in view of the rational
processes (photolithography, wet or dry etching methods etc.) for fabrication, which
can be used to obtain a large number of identical functional microstructures positioned
at highly defined coordinates [16]. In order to produce cheap disposable devices in
other materials than glass or silicon, polymer replicas have been introduced [19, 20].
The first micro/miniaturized total analysis system (µTAS) on a silicon chip, was a
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Erik Litborn
micro gas chromatograph [43] that was presented in the late seventies. In 1992,
Harrison and Manz et al. introduced the Lab-on-a-Chip concept [44, 45] with a chipbased device for capillary electrophoresis. Since then, many different devices have
been presented in the literature and at conferences e.g. µTAS3. Commercial chip-based
devices are now also available [46]. Stacked devices have been presented where layers
are connected to each other in order to construct a more complete µTAS device [47,
48].
Most of the µTAS chips have been designed for chip based capillary electrophoresis
and an overview of the development within this area can be found in the following
review articles [17, 49-52]. However, only a few of these devices have a reactor
included [53-62]. Other application areas for µTAS chips are e.g. capillary
electrochromatography (CEC) [63] with packed reactor beds and immobilized
reagents or systems combined with electrospray mass spectrometry [64-70], cell
handling [71-76] and immunosorbent assays [77]. PCR reactors [78-80] without
electrophoretic separation of the amplified DNA have also been presented. The SVR
in these chip-based systems allows efficient and rapid heating and cooling during the
PCR thermo cycling.
A major drawback with chip-based devices is the difficulty of interfacing the real
world and the miniaturized lab. The microchips need to be integrated in a system with
both sample preparation and analysis in order to accomplish the potentional savings of
volumetric reduction [13]. Also, as for the capillary based reactors, the sample transfer
and handling procedures usually requires µL sized volumes of solutions, even though
a reaction can be carried out in nL – pL sized volumes. The integration of different
components onto a chip is not trivial [81] but once the sample is introduced into the
chip device, electrokinetic control of the fluid flow [82-85] enables the handling of pL
sized volumes of sample [86]. Not only water based fluids can be electrokinetically
controlled; organic solvents can be handled as well [48, 87, 88]. Distribution of fluids
and reagents using centrifugal forces is a different approach for sample handling in
chip-base devices. This has led to the development of a device in a chip-based format
very similar to a music compact disc (CD) [89]. Also, pneumatic pumping of pL
volumes of reagents has been presented using hydrophobic microcapillary vents
fabricated in poly(dimethylsiloxane) (PDMS) [90]. Pneumatic as well as hydrostatic
pumping has been used in a microfabricated device of PDMS used for dynamic DNA
hybridization using paramagnetic beads [91].
3
http://www.mesaplus.utwente.nl/mutas2000/
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Sample Handling in Nanoscale Chemistry
2.2 Open systems
2.2.1 Surface based reactors
The simplest form of an open miniaturized reactor is to deposit drops of reagents onto
a surface with pre-immobilized chemicals to perform an assay [92]. Devices have been
produced e.g. gene-chips for DNA hybridization [93, 94] where oligonucleotides are
synthesized in situ using a photolithographic technique. The resulting chip contains all
combinations of an octanucleotide in a high-density array. Alternative manufacturing
methods for DNA chips are electrodirected copolymerization of pyrrol [95], where
covalently attached primers or templates are dispensed onto a functionalized gold
surface [96, 97] and immobilization of oligonucleotides within miniature gel-pads
arranged on a microchip [98, 99]. For protein characterization, trypsin immobilization
directly onto a matrix assisted laser desorption ionization (MALDI) target can be used
to produce a surface based reactor [100]. Patterns of proteins have also been produced
by microcontact printing using PDMS stamps [101]. The surface based reactor format
is very useful for performing assays where the test solution is in contact with several
reactors at the same time. Such reactors have been used for growing cells (different
cells are regarded as different reactors) in a common medium [7] for phenotypic
macroarray analysis.
2.2.2 Chip based reactors
For a reaction that requires a long reaction time in free solution, the use of open micro
reactors or vials is very useful. Some of the attractive features are:
• A large number of individual reactors (as many as 500,000) can easily be
manufactured on a single chip (glass, silicon, quartz or plastic).
• Random access to individual vials is possible, using a precision robotics
system. Moreover, open systems can be accessed any time before, during and
after a reaction (for e.g. addition of reagents, withdrawal of sample or
detection).
• The sample is kept in a confined space. Sample dispersion and/or dilution,
which is an inherent problem in continuous flow systems, is avoided.
• No carry-over problems are experienced, as is due to dispersion and lagging
(adsorption) of sample in sequential flow reactions.
However, there are also drawbacks of the open vial concept, notably:
• Contamination from the outer environment (dust, including biological
material) can be a severe problem.
• When dealing with a large number of different samples, the transfer of small
sample volumes demands relatively complicated systems, such as arrays of
micro-capillaries. However, this sample transfer problem is not trivial in flow
systems either.
• Evaporation of the reaction solvent.
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Erik Litborn
The concept of miniaturized open chip-based nanovials was introduced in 1992 by
Jansson et al. [102]. The nanovials were used as sample containers for electrokinetic
injection of DNA samples prior to a CE separation. By using photolithography and
anisotropic etching techniques, a large number of such vials could be manufactured on
a monocrystalline silicon wafer, Figure 1. Later on, the usefulness of the open chipbased vials, with nano- and picoliter sized volumes, was demonstrated as targets for
MALDI-TOF MS analysis of proteins (Paper I), and as reaction vessels for tryptic
digests of myoglobin followed by an electrophoretic separation of the obtained
peptide map (Paper III, IV&V).
L
L
Figure 1: A schematic of the chip-based vials used. The manufacturing procedures are 1:
transfer of a pattern from a photomask to a monocrystalline oxidized silicon wafer by
using photoresist and photolithography, 2: development of the photoresist pattern and
creation of an etching mask (SiO 2), 3: anisotropic etch of the wafer, 4: deposition of a thin
conductive film of gold to enable an electrical contact during electrokinetic injections or
MALDI-TOF MS analysis. The vials have an inverted pyramidal shape with the angle
54.74° between the flat surface and the vial wall. The vials used have side lengths ranging
from 50 - 400 µm (corresponding volumes: 30 pL - 15 nL).
The concept of using open nanovials has also been taken up by several other workers
for electrochemical studies [103-108], bioluminescence measurements [109] and as
MALDI-TOF MS targets for analysis of oligonucleotides [110], peptides [111, 112]
and organic molecules [113]. Fabrication technologies that have been employed to
produce the vials are etching [103, 104, 110], laser ablation [108, 109], replica
imprinting from micromachined masters [105-107], as well as etching optical fibers
[114, 115].
The trend towards miniaturization is very clear. Although, the 96-well plate is still the
most commonly used format for open reactors, systems for e.g. PCR [116, 117] have
been miniaturized from the 96-well format to an 864-well format [118] and screening
assays have been miniaturized (200 nL vial volume), using a plate design with 9600
wells in each plate [8]. Other non-standard miniaturized formats have been presented
where molding techniques have been used for producing wells in
polydimethyldisiloxane and polypropylene (2304 wells of 8 µL each) [119, 120].
Drilling has also been used to produce arrays of nanowells in acrylnitrile buthadiene
styrene (ABS) plastic sheets (2025 wells of 0.37 µL each) [5].
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Sample Handling in Nanoscale Chemistry
3. Dosing of liquids into open vials
There is a rapidly growing need in analytical chemistry for devices, which can
dispense small volumes of fluid. It is very difficult to handle sample volumes in the nL
– pL range using conventional pipettes or syringes which are commonly employed in
most automated systems. The tendency of small droplets to adhere to surfaces also
increases the difficulties of dosing when the volumes are reduced. In this section,
different strategies for dosing small volumes of liquid are divided in contact and noncontact principles. Depending on the application, combinations of the presented
principles might be advantageous to utilize.
3.1 Contact dosing
In the early development of equipment for dispensing, most devices were based on the
use of syringes. One of the drawbacks with the use of syringes is their relatively large
dead volumes. In order to reduce the volumes needed to dispense reagents, devices
based on miniaturized pipette tips (Paper I, III & IV) or pressurized capillaries
(Paper IV &V) can be used. For a detailed description of the dosing principle using
capillaries, see section 4.2.4.1. The smallest dead volume can be obtained when
miniaturized pipette tips are used, but the necessary flow regulation is more difficult to
achieve compared to the use of a capillary. This is (probably) due to an increased
pressure drop caused by the small dimensions of the drawn tip. Filling of the tip is
performed by capillary action through the nozzle of the tip. Dosing is performed by
applying an overpressure to the tip while it is positioned inside a well or a vial. The
dosed reagent will form a droplet attached to the tip until contact with the wall of the
well (or a liquid inside the well) is established. Commercial pressure driven
micropipettes are available and have been used for dosing 10 pL sized volumes [121].
Contact dosing can be performed by using a tip with a slot (compare the action of an
old fashioned ink pen), which soaks in a certain volume of liquid by surface tension
when it is dipped into a solution. When the tip is brought in contact with a solid
surface, a reproducible volume of the reagent is dispensed onto the surface [122]. This
technique is only suitable for surface based reactors and is not so useful for dosing a
reagent into an open microvial. However, if the bottom of a vial is flat, contact dosing
of a small reagent volume should be possible to perform. A complete description of
how to build your own “micro-array printer” can be found on the Brown Lab’s
homepage4.
An important drawback with the different contact-mode dispension techniques is the
risk for cross-contamination.
4
http://cmgm.stanford.edu/pbrown/mguide/index.html
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Erik Litborn
3.2 Non-contact dosing
Methods for non-contact dosing can be divided according to the driving force utilized.
Electrospray deposition (ESD) has been proposed for dosing reagents [123, 124] onto
surfaces. The devices are very similar to the nanospray tips [125] commonly used for
sample introduction in electrospray ionization mass spectrometry. The amount of
deposited material is determined by weight using a quartz microbalance [123]. Dosing
is performed by applying a potential between the spray nozzle and the target and thus
creating a local electrostatic field. An interesting feature is that parallel dosing can be
performed using a dielectric mask that addresses the reagents to predetermined
positions (defined by the mask) [124]. ESD is also useful for sample preparation in
MALDI-MS due to the formation of very fine crystals of the MALDI matrix [126,
127].
Non-contact dispensing can also be performed by applying a pressure pulse to a
confined volume of solution, allowing an aliquot of the solution to leave the larger
bulk volume via a small exit hole. A fast pressure increase of the pulse is needed to
overcome surface tension forces and to expel a small droplet of the solvent. The pulse
can be created by a solenoid valve mounted between the exit hole and the bulk
volume. This technique is preferable to use for dispensing of µL-sized volumes [120]
but can be used for volumes down to 100 nL5.
The pressure pulse can also be created by using piezo-ceramic materials. Devices for
controlled piezo-electric dosing of nL - pL sized volumes of chemicals are now being
developed at rapid pace due to the presence of a well-developed technology of inkjets, commonly used in high-resolution printers. Great precision can be obtained and
the usefulness of piezo-electric dispensers has already been demonstrated in different
applications [110, 128-131]. The piezo-electric dispenser utilized in Paper II for
performing nanoliter titration [129] employs a glass capillary tube with a constricted
outlet. A glued-on piezo-ceramic body surrounds the capillary tube. When a voltage
pulse is applied to the body, a contraction occurs, which generates a steep pressure
pulse within the capillary. If the capillary is filled with liquid, a small portion of the
liquid will discharge from the outlet. Under optimized conditions, the expelled liquid
can form a single droplet with an almost perfect spherical shape. The piezo-ceramic
material can also be positioned at one side of a flow channel with the dispensing
nozzle on the opposite side [132, 133]. Also, dispensers using flat chambers with the
piezo-electric material mounted on one side, perpendicular to the nozzle, are
available6.
Droplet formation during piezo-electric dispensing is governed by a number of interdependent parameters, such as the nature (viscosity, surface tension) of the liquid, the
shape and size of the nozzle, the characteristics of the applied electric pulse (voltage
and pulse length), etc. Frequently, small satellite droplets are formed along with the
5
6
Gesellschaft für Mikrodosiersysteme mbH, http://www.microdrop.de
Gesellschaft für Silizium -Mikrosysteme mbH (GeSiM), http://www.gesim.de/
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Sample Handling in Nanoscale Chemistry
main droplet, which is detrimental for the dosing precision. However, it is rather
straightforward to obtain single droplets by adjusting the voltage and the duration of
the electric pulses sent to the piezo-ceramic body. In this context, it is important that
the nozzle of the dispenser is clean and not wetted by the solvent. For dispensing
aqueous solutions, we found that silanization of the dispenser tip is necessary.
However, organic solvents such as iso-propanol are very difficult to dispense, even
with silanized tips. An initial experiment with a surface coating using a perfluorinated
compound has shown promising results.
3.3 Piezo-electric nanoliter titration (Paper II)
A controlled and incremental addition of solutions from a piezo-electric dispenser can
be utilized for determination of the absolute amount of a substance by titration. Hieftje
et al. constructed a device, which made it possible to generate a continuous stream of
electrically charged reagent droplets of ca 350 nL volume [134]. Individual droplets
were directed into a titration vessel, by applying an electric field. One major drawback
of this procedure is the need for a continuous supply of titrant, which results in large
waste volumes. Another device for the generation of smaller droplets was developed
using a vibrating glass needle [135]. In this way, a continuous stream of small droplets,
having volumes down to ca 3 nL, was generated. However, a generation of single
droplets on demand was not possible, and therefore the minimum volume, which
could be dispensed with accuracy, was ca 40 nL [136, 137]. We have used the piezoelectric dispenser to perform automated titrations of low nL analyte volumes (Paper
II). The incremental addition of titrant is easy to control by a drop-on-demand (DOD)
addition. The system includes a detection system, based on wave-guided laser-induced
fluorescence [138]. The use of precision robotics makes it possible to rapidly perform
a large number of titrations in a consecutive order. The titration set-up is evaluated by
performing repetitive fluorometric titrations of ca 9 nL of H2SO4 with NaOH.
A correct calibration procedure of the system is very important. This can be achieved
“on-the-fly” by determining the volume of the droplets by measuring their diameter
[139-141] e.g. by using a microscope and image analysis. However, to obtain high
precision data is not straightforward due to difficulties in defining boundaries of curveshaped objects, like droplets or fibers, the latter can be used as calibrating standards
for measurements with a charged coupled device (CCD) camera. Moreover, errors in
observed droplet diameter propagate to the third power in errors of volume. An
alternative method would be to dispense a number of droplets on a microbalance.
From the weight of the dispensed material, the volume of an individual droplet could
be calculated, if the density of the solution is known. However, weighing procedures
also inherit certain disadvantages. The density of a solution is not always available
when dealing with unknown samples and volume determination by weighing will be
affected by the evaporation of solvent during the time between dosing and weighing.
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Erik Litborn
A titration is started by pooling a predetermined number of sample droplets (including
the indicator), on the surface of a polyethylene support using a first piezo-electric
dispenser. Next, the pooled sample is repositioned, using an X-Y table, under the
nozzle of a second piezo-electric dispenser filled with reagent. The titration process is
started by discharging single droplets of reagent into the sample at a predetermined
rate and simultaneously, the data collection from the fluorescence detection is started.
After a preset number of titrant droplets have been added, data collection is
discontinued and the collected titration data are exported to a PC to perform the endpoint evaluation. Automated repetitive titrations (100 titrations) are performed by
repeating the entire procedure but at a new individual titration position, determined by
the programmed control unit.
Figure 2 shows the first results from automated fluorimetric piezo-electric titrations.
The calculated volume of titrant, necessary to reach the equilibrium point is
represented by the height of the vertical lines for the corresponding samples. Initially
(for the first 30 titrations), the reproducibility was satisfactory, while a substantial
deterioration was observed for the subsequent titrations. We found this problem to be
related to a wetting of the nozzle and the problem was solved by treating the tip with
Repel-Silane. The effect of the treatment lasted for about one working day. The greatly
improved results are shown in Figure 3. The excellent reproducibilities obtained for
different titrations are presented in Table 1 and Table 2.
25
nL titrant added
20
15
10
5
391
381
371
361
351
341
331
321
311
301
291
281
271
261
251
241
231
221
211
201
191
181
171
161
151
141
131
121
111
91
101
81
71
61
51
41
31
21
1
11
0
Sample number
Figure 2: Results from titrations performed with non-silanized piezo-electric dispensers.
Sample: 10 drops (each drop with a volume of 0.90 nL 50 mM H2SO4), Titrant: 80 drops
(each drop with a volume of 0.52 nL, 50 mM NaOH). Time between each added droplet of
NaOH: 1000 ms for sample 1-100, 500 ms for sample 101-200, 250 ms for sample 201300 and 3000 ms for sample 301-400. Calculated volume of added titrant: 18.1 nL.
- 10 -
Sample Handling in Nanoscale Chemistry
40
+
35
+
nL titrant added
30
+
25
20
15
10
5
295
285
275
265
255
245
235
225
215
205
197
187
177
167
157
147
137
127
117
99
107
89
79
69
49
59
39
29
9
19
0
Sample number
Figure 3: Three sequences of consecutive titrations performed at different titration rates.
The tips of the piezo-electric dispensers were treated with Repel-Silane before starting the
measurements. Sample: 10 drops (each drop with a volume of 0.90 nL 50 mM H2SO4),
Titrant: 80 drops (each drop with a volume of 0.52 nL, 50 mM NaOH). Time between each
droplet: sample # 1 – 100: 1000 ms, sample # 101-200: 500 ms, Sample # 201-300: 250
ms. Calculated volume of added titrant to reach the equilibrium point: 18.1 nL. Note the
anomalous results of sample 1, 101, and 201 (marked with +), which is due to initial
evaporation of water from the sample in the piezo-electric dispenser tip.
Table 1: Calculated average titration volumes with standard deviations for titrations
performed at different titration rates. The results are compared with the calculated titration
volume (18.1 nL NaOH).
Titration rate –
time between droplet addition
(ms)
250
500
1000
2000
3000
Average –
first ten titrations
(nL NaOH)
15.9
16.6
17.1
16.5
18.1
- 11 -
Stdev –
first ten titrations
(%)
1.5
1.4
0.78
0.86
1.7
Erik Litborn
Table 2: Calculated standard deviations for titrations performed with the same titration
rate (1000 ms between droplet addition) at different concentration levels. The standard
deviation of the results obtained at the lowest concentrations (marked *), is calculated only
from three titrations.
Concentration OH(mM)
50
5
0.5
0.05
Concentration H+
(mM)
100
10
1
0.1
- 12 -
Stdev – first ten titrations
(%)
0.78
1.03
1.23
1.63*
Sample Handling in Nanoscale Chemistry
4. Reactions in open chip-based vials
Miniaturized systems for performing reactions can offer many advantages, such as
reduced costs for reagents, possibilities to maintain high reaction concentrations,
possibilities to perform chemistry when a limited amount of sample is available etc. In
the work with open chip-based vials, we have chosen enzymatic degradation of
proteins as a model application since there is an increasing need for tools and methods
within e.g. proteomics where many proteins are available only in limited amounts.
Normally, proteins are denatured prior enzymatic degradation in order to open up the
structure and thus exposing the cleavage sites. This results in an increased conversion
rate from protein to peptides. We have chosen native myoglobin as a substrate since
the globular structure often requires long digestion times. Native myoglobin can
therefore be regarded as a suitable model substance to use in a general method
development for digests.
Controlled enzymatic digestion of proteins (or peptides) is one of the standard
procedures in biochemistry [142]. In summary, a proteolytic enzyme is added to the
protein in a buffer solution. During the incubation (digestion), the enzyme cleaves the
protein at cleavage sites typical for the enzyme used. From a specific fragment, the
order of the amino acids can be determined in further investigations using Edman
degradation [142]. The use of several assays based on enzymes with different cleavage
sites is advantageous. In many cases, peptide fragments produced (using e.g. trypsin)
contains specific cleavage sites typical for a second enzyme (e.g. endoproteinase GluC) and vice versa. Thus, many of the fragments can be correlated to each other using
the determined overlapping amino acid sequences. This results in valuable information
of the amino acid sequence for structure elucidation of the entire protein.
The most frequently used enzyme for performing digests is trypsin with cleavage sites
on the carboxylic side of the amino acids lysine and arginine in the chain of amino
acids. After the digest, the peptide mixture can be analyzed by liquid chromatography
[143, 144], capillary electrophoresis (CE) [145], electrospray-MS [146] or matrix
assisted laser desorption/ionization mass spectrometry (MALDI-MS) [147, 148]. The
result of the analysis is presented as a peptide map (a “fingerprint”) of the investigated
protein. Such peptide maps are very useful for determination and characterization of
primary structures of proteins. Protein digest-protocols are also often used in purity
verification and control of precision and reproducibility in recombinant processes.
Comparison between similar proteins can reveal minor structural differences.
Moreover, preliminary identification of proteins can be performed by comparing
peptide maps with maps of known substances.
A fundamental problem of the open vial concept is the evaporation of solvent (water)
from the vials. Without any countermeasure to prevent or reduce evaporation, nL - pL
sized volumes evaporate in a few seconds when left in the ordinary atmosphere.
Several strategies have been proposed for dealing with this problem but the use of one
single method is not always suitable for all different situations.
- 13 -
Erik Litborn
4.1 Nanorobot
Due to the requirements to precisely position tools and other equipment needed for
adding reagents, for performing reactions and to carry out subsequent analysis, a
computer-controlled robot was constructed. Figure 4 shows a schematic of the robot
used in the work with parallel-operated reactions (Paper IV).
End of the CE capillary connected
to buffer container (anode)
To pressure regulation
CE separation capillary
GND (CE cathode)
Micropositioner
Outlet
Copper holder
Inlet
Fused silica capillary tips
for delivery of reagents
Tubing for CE buffer
replenishment
DC voltage to Peltier Element
Peltier element
Silicon chip with arrays of vials
Perspex holder with
alignment grooves
for 8 capillaries
Running buffer container
To pressure regulation
XY-table
Solvent for compensation
of the evaporation
XYZ micro manipulator
Figure 4: A schematic top-view of the nanorobot constructed for performing
nanochemistry. It consists of a computer controlled XY-table with 1 µm addressable
precision. Several vertical mounted micro-positioners are used for positioning along the zaxis. The current schematic shows the set-up utilized for performing parallel reactions in
15 nL sized vial volumes (Paper IV). Depending on the application, different tools are
mounted onto the positioners e.g. a cutting wheel for capillaries (Paper IV), piezo-electric
dispensers (Paper II), optical fibers (Paper VI), solvent supplying capillaries or capillary
tips (PaperIV&V), a CE separation capillary (Paper IV&V). Since the XY-platform is
rather large (20*20 cm), a manual XYZ-manipulator can be mounted onto the XY-table
together with different containers or chip-holders. All operations are observed using a long
distance working microscope equipped with a CCD camera and an image analysis system.
- 14 -
Sample Handling in Nanoscale Chemistry
4.2 Methods for control of evaporation of water
4.2.1 Use of mechanical lids
The most common method to prevent evaporation is to cover the reaction mixture
with a solid or mechanical lid [119, 149]. Plastic chips can be effectively sealed by
welding a thin foil of plastic over the wells [150]. This is straightforward for µL sized
volumes but not easy to apply on nL–pL sized volumes. A problem that typically
occurs is that part of the reaction mixture adheres to the lid.
A very innovative application of a solid lid of gold covering a reservoir has also been
presented [151]. The lid was used to control the release of a component into a mixture
by dissolving the lid electrochemically but has not been used for covering a reactor
during incubation.
4.2.2 Addition of non-volatile matrices
Evaporation can be reduced by addition of non-volatile matrixes to the reaction
mixture, Figure 5, which reduces the vapor pressure of the solvent and thus reduces
the driving force for evaporation (see section 4.2.4.2). This has successfully been used
by Ewing et al. and others [104, 106, 119] for nL sized reaction volumes in open vials.
Matrix ( ) added
to the reaction mixture
Figure 5: The vapor pressure is reduced by adding a high-boiling matrix to the reaction
mixture.
However, when reactions are carried out under an extended period, an additional
humidified environment seems to be necessary. The main drawback of matrix addition
is a possible interference of the additives with the reaction and its analytes. In addition,
the additives may be detrimental contaminants in subsequent analytical measurements
(such as CE separations, mass spectrometry etc.).
- 15 -
Erik Litborn
4.2.3 Increase of humidity (Paper III)
Another approach to reduce the evaporation is to enclose the sample in a humidified
environment [8]. In principle, working at 100% relative humidity would completely
prevent an evaporation of water from the sample, Figure 6.
Environment with
increased humidity
Reaction mixture
Chip with reaction vial
Enclosure
Water-bath
Figure 6: The evaporation of water from an open vial can be reduced by increasing the
humidity in the surrounding environment.
However, operation at this humidity level is not possible without introducing severe
problems. At a very high humidity level, water starts to condense onto irregularly
shaped surface areas, like scratches, dust etc., leading to overflow to adjacent vials and
cross contamination. The behavior is related to differences in surface wettability
(contact angle), the strive of surfaces to minimize their free energy and the associated
well-known phenomenon of capillary condensation [152]. Another problem is that the
evaporation rate of water from a sample is affected by the presence of salts and other
components, which can cause an excessive condensation of water into the sample
vials. In practice, the concept is useful if the humidity is kept at a level where
condensation does not occur, but a periodic water addition is then necessary (Paper
III). Operation becomes increasingly difficult at elevated temperatures.
In Figure 7, the result from a tryptic digest of native myoglobin operated in a
humidified environment and performed in a nanovial (15 nL) is compared with a
digest performed in a conventional plastic vial (100 µL). Considerable differences can
be seen between the electropherograms but the mid regions show a notable similarity.
The performance of the digest (the rate of protein conversion into peptides) seems to
be dependent on the type of vial used. This can be measured as the ratio between the
height of a peak representing a peptide (A or B) and the original myoglobin peak
(myo). The peak ratio is higher when the digest is performed in a nano-vial compared
to a digest performed in a conventional plastic vial. It is suggested that this is the result
of the higher SVR in the nanovial, which is further discussed in 4.3.
- 16 -
Sample Handling in Nanoscale Chemistry
my
o
my
o
A
B
Figure 7: Two electropherograms obtained after CE analysis from tryptic digests of native
myoglobin performed in a chip-based nanovial (left) and in a conventional plastic vial
(right). The conversion from protein to peptides can roughly be calculated from the ratio
between the peak height of a representative peptide clearly notable in both
electropherograms (A and B) and the peak height of the remaining undigested myoglobin
(myo).
4.2.4 Continuous compensation (Paper IV)
Instead of reducing the rate of evaporation, another principle can be applied, where
water is added continuously to the vials to compensate for evaporation (Paper IV).
When a continuous supply of solvent (e.g. via capillaries, connected with the reaction
vials and a solvent supply vessel) is kept in balance with the evaporation, reactions can
be sustained over many hours or even days. However, it is important to utilize very
pure solvent, since impurities from the solvent are accumulated in the sample.
Continuous water addition
via a capillary
Figure 8: The amount of evaporated solvent from a nanovial is continuously compensated
for. The other end of the capillary is connected to a solvent supply vessel (not shown in the
figure).
- 17 -
Erik Litborn
Continuous compensation of water can be performed in parallel by arranging the
solvent feeding capillaries in a holder with V-shaped grooves. Figure 9 shows the
result of the CE-analysis of the content in eight separate vials wherein enzymatic
degradations of myoglobin have been performed.
T
T
C
C
C
G
G
G
Figure 9: Eight electropherograms obtained after the CE analysis of eight individual
enzymatic digests of native myoglobin (7 pmol in each vial) performed in parallel in chipbased vials. The electropherograms are labeled according to the enzymes used: trypsin (T),
α-chymotrypsin (C) or endoproteinase Glu-C (G).
Reaction conditions: chip-based vials with 15 nL volumes, 8 mg/mL myoglobin, 8 mM
NH4HCO 3 pH 7.9, 0.2 mg/mL enzyme concentration, 37 °C, reaction time 4 hrs (prior
analysis of the content in the first vial).
CE conditions: separation capillary ID 50 µm, OD 150 µm, total length 102 cm (72 cm to
the detector), 0.01 M phosphate pH 7.0, 100 µg/mL FC 134, +15 kV 5 µA.
- 18 -
Sample Handling in Nanoscale Chemistry
We have also constructed an array of 32 solvent feeding capillaries with dimensions,
suitable for parallel reactions in 1 nL chip-vials, Figure 10.
Figure 10: A photograph showing a nanodispenser fitting 1 nL chip-based vials. The size of
each individual vial is 200*200 µm and the outer diameter of the capillaries is ca 70 µm.
The width of the entire dispenser is ca 15 mm.
This device is manufactured by precise positioning of the feeding capillaries using a
holder with V-shaped grooves matching the spacing between the chip-based vials. The
holder is a plastic replica made from a micromachined master. After gluing the
capillaries in position, a small scratch is made on all the capillaries in order to enable
careful breaking of the capillaries [153]. A cutting wheel (tungsten carbide), normally
used for cutting optical glass fibers, was utilized for this purpose. The fabrication of
larger arrays as well as capillaries, designed for still smaller reactor volumes, should be
quite feasible in this way.
4.2.4.1 Filling procedure
The filling procedure of a vial starts with applying a very low overpressure in the
solvent vessel in order to form a small solvent meniscus at the end of the capillary,
Figure 11A. When the meniscus is brought in contact with the vial wall, the surface
tension is broken and a flow of solvent starts to fill the vial, Figure 11B.
Simultaneously, the capillary is moved upwards until the solvent level inside the vial
has reached a level where the evaporation from the solvent surface counterbalances
the flow. In this respect, the inverted pyramidal vial has a beneficial shape since the
surface area of the solvent meniscus increases rapidly as the volume of the liquid
increases. Due to this increase in surface area, the evaporation also increases rapidly to
a point where it counterbalances the flow from the capillary. The vial can even be
overfilled without overflow (c.f. “topping” a glass of water), Figure 11C. This is due to
- 19 -
Erik Litborn
the effect of the surface tension and the abrupt change of angle between the sidewall
of the vial and the horizontal surface. When the vial is completely filled, a steady state
can be maintained for several hours with the solvent supply in balance with the
evaporation (Figure 11D).
Overpressure
Withdrawal of capillary Steady state
Filling initiated while filling the vial
(continuous solvent addition)
Vial wall
Fluid flow
A
B
Evaporation
C
D
Figure 11: Schematic of the procedure of filling a vial. The schematic only shows the
capillary end that is in contact with the vial. The other end is connected to a solvent supply
vessel not shown in the figure. A: An enlargement of the end of the solvent supplying
capillary showing the formation of an initial solvent meniscus caused by overpressure in
the solvent supply vessel. B: When the liquid touches the vial surface, the surface tension of
the meniscus is broken and the filling of the vial is initiated. C: The solvent supplying
capillary is moved upwards, while filling the vial. D: The evaporation of solvent from the
vial is counter-balanced by a continuous flow of solvent through the capillary. N.B. The
capillary is withdrawn from the vial after event C, in the case where a reagent solution is
added to a vial.
4.2.4.2 Evaporation theory for continuous compensation
The driving force for the evaporation of solvents into air is the difference between the
vapor pressure of the solvent just above the surface and the vapor pressure in the bulk
air. If the air is slowly passed over the surface, a boundary layer is formed with a
concentration gradient of the solvent within it. Since the dominating resistance against
mass transport is in the diffusion boundary layer [154], focus will only be given on
this.
The diffusivity of vapors in different gases, from a planar surface to a gas stream, can
conveniently be determined with a method developed by Winkelmann [155]. In short,
the method is based on the formation of a boundary layer (using a controlled diffusion
distance and thus with a known thickness of the layer) between a solvent vessel and a
stream of dry air or gas. The amount of evaporated material is straightforward to
measure when large volumes of solvent (several mL) are used, and therefore the
evaporation rate can easily be calculated. However, the method is not straightforward
to use for the determination of evaporation rates in the range of nL/min since small
- 20 -
Sample Handling in Nanoscale Chemistry
volume changes are difficult to measure. In addition, the thickness of the boundary
layer formed is difficult to determine when the evaporation of solvents to stagnant air
is to be examined (the diffusion distance is not exactly defined). To circumvent these
difficulties, we have based our calculations on a mass transfer theory, where the shape
of the solvent surface is defined as a hemisphere. In order to be able to apply this
theory in a straightforward way, the surface area of the square entrance of our vials
was recalculated to the surface area of half a droplet with a corresponding surface area.
Mass transfer from a single water droplet to stagnant air follows the law of molecular
diffusion. The rate of evaporation (W) can be calculated from the diffusivity
(temperature dependent) of water in air (D), the surface area of the droplet (4π r2), and
the difference of humidity (d(κ)), at a given distance from the surface (dr) [154]:
W = − D * 4π r 2 *
d (κ )
dr
(1)
After integration from the radius of the droplet (r1) over the distance out to the bulk air
(at the distance r2) with the corresponding temperature dependent vapor pressure over
the surface (κ1) and the vapor pressure in the bulk air (κ2), the expression results in:
r2
κ2
1
1
dr
W ∫ 2 = −4 π D ∫ d κ
r r
κ
W =
4π D (κ 1 − κ 2 )
(1 − 1 )
r1
r2
r ⇒∞
Since the thickness of the diffusion boundary layer (r2) is infinitive, the expression
results in eq (2).
r2 ⇒ ∞
W
=1
4π rD (κ 1 − κ 2 )
(2)
The evaporation rate of solvent from a liquid surface to the surrounding bulk air can
also be expressed as the mass transfer coefficient (hD) [154],
hD =
W
4π r (κ 1 − κ 2 )
2
2r = d drop
- 21 -
(3)
(4)
Erik Litborn
where r is the radius and ddrop is the droplet diameter. After substitution of eq (3) and
eq (4) into eq (2), the mass transfer coefficient, the diameter of the drop and the
diffusivity can be related to each other according to eq (5).
hD d drop
D
= 2 = Sh
(5)
This expression is called the Sherwood number (Sh) [154]. For a spherical droplet, the
Sherwood number is always equal to two and describes the mass transport from the
droplet into stagnant air. In our case, we regard the liquid surface area of a square vial
as a corresponding half droplet. The droplet diameter used (ddrop) is calculated from a
droplet which half area (2π r2) with the same area (A) as the planar liquid surface in the
vial. The evaporation rate of solvent from a vial at different temperatures can now be
calculated utilizing the calculated, eq (5), mass transfer coefficient (hD), the area of the
vial (A=L*L, Figure 1), and the difference of vapor pressure, (κ1-κ2):
W = hd * A * (κ 1 − κ 2 )
(6)
4.2.4.3 Evaporation measurements during continuous compensation
Evaporation rate (nL/min)
During the reaction, a large amount of water is evaporated from the vials. The amount
can be measured using two different methods (Paper IV). The first method (M1) is
based on measuring the time for one vial volume of solvent to evaporate and the other
method (M2) is based on measuring the flow of solvent in a solvent feeding capillary.
In Figure 12, the measured evaporation rates obtained for different solvents (water,
cyclohexane and ethanol) in a chip-based 15 nL sized vial are obtained. The rates are
measured with the two methods and are compared with the calculated theoretical
values using equation (6).
900
Water (M1)
800
Water (M2)
700
Water (theo)
600
Cyclohexane (M1)
500
Cyclohexane (M2)
400
300
Cyclohexane (theo)
200
Ethanol (M1)
100
Ethanol (M2)
0
Ethanol (theo)
10
20
30
40
50
60
70
Temp (C)
Figure 12: Measured evaporation rates for water, cyclohexane and ethanol using methods
based on measuring the time for one vial volume to evaporate (M1) and the flow in the
solvent feeding capillary (M2) compared with theoretical values (theo) calculated using
equation (6).
- 22 -
Sample Handling in Nanoscale Chemistry
Turn-over rate
(vial volumes/min)
In Figure 13, the turnover rates (number of vial volumes of water evaporated) for
water evaporated from vial volumes (15 nL – 30 pL) at different temperatures are
presented. The evaporation rates were measured using the method that was based on
measuring the solvent flow in the feeding capillary (M2).
140
120
100
80
60
40
20
0
10
20
30
40
50
60
70
Temp (C)
Figure 13: The turnover rates (number of vial volumes evaporated over a period of time)
for water at different temperatures for vial of different volumes (30 pL – 15 nL).
4.2.5 Use of liquid lid (Paper V&VI)
Mineral oil
Figure 14: The evaporation can be prevented by a complete coverage of mineral oil over
the entire chip (left) or in each individual vial (right).
For applications that require high temperatures, such as PCR, evaporation can be
eliminated by covering the reaction mixture with a non-volatile liquid lid e.g. mineral
oil [103]. The oil can cover the entire chip or be added separately to each vial, Figure
14. However, the drawback with mineral oil appears when the lid is to be removed.
This is quite difficult since mineral oil tends to stick to most surfaces and residual oil
will remain present. Instead, an attractive solution is to cover the reaction liquid with a
film of a non-miscible volatile liquid. This method was first utilized by Gratzl et al.
[141] who carried out diffusional titrations of pL-sized samples, which were
submerged in heptane in a Petri-dish.
- 23 -
Erik Litborn
Evaporation of water from solutions kept in chip-based vials has been prevented by
creating a thin layer (0.5-1 mm) of a volatile non-miscible liquid over the chip
(Paper V). In order to prevent a premature evaporation of the cover liquid, a
continuous supply of the liquid is guided over the surface. Figure 15 shows a
schematic of the principle.
Evaporation of the covering liquid
Drainage hole
Inlet of the volatile
covering solvent
Outlet
Figure 15: The principle of preventing evaporation of water from the chip-based vials by a
complete coverage with a volatile liquid lid. Consequently, solvent has to be continuously
added to the lid in order to compensate for the evaporation.
The advantage of this setup is that a similar degree of accessibility as in open vials is
obtained, while the vials are always sealed by the liquid lid. Reagents can be added or
sample can be withdrawn any time during or after a reaction, by guiding
robot-controlled micropipettes or the inlet of a CE-column through the covering film
close to or into the sample. Contamination of the pipettes or the CE column by
adhered covering liquid is not a problem, since this liquid evaporates as soon as the
pipette or the CE column is withdrawn, leaving a clean sample exposed to the
environment. Moreover, the entire liquid lid (and also the solvent of the sample) will
evaporate completely, when the supply of covering liquid is discontinued.
However, some losses of reaction solvent were noticed after an extended period of
operation. This has been investigated by observing a chip-based vial filled with 15 nL
of water, kept at 37°C covered with a thin, flowing film of octane (estimated film
thickness, ca 0.5 mm). The meniscus of the water sample was monitored by using the
microscope and the video camera of the robotics equipment. The results are shown in
Figure 16 (left). After 90 min, no loss of water was observed. After 105 min, the first
losses were clearly visible, and after 140 min, losses are on the order of 50%. The
experiments were also repeated with water-saturated octane, in order to compensate
for possible effects of water dissolution, however no improvement was observed. In
addition, a significant dependence of the flow rate of octane on the loss of water was
not observed. It seems likely that the water loss occurs primarily via diffusion through
the thin layer of the covering solvent.
- 24 -
Sample Handling in Nanoscale Chemistry
0 min, 37°C
1 min, 95°C
105 min, 37°C
3 min, 95°C
120 min, 37°C
5 min, 95°C
140 min, 37°C
Figure 16: Photographs showing the loss of water from a 15 nL chip-based nanovial under
different conditions (see text).
Further, experiments at higher temperatures were also carried out. It showed to be
possible to retain most of the water sample in the vial at a temperature close to the
boiling point (95°C) during several minutes (Figure 16 right). This performance is
unique in the sense that is well beyond the temperature range of the other noncontaminating methods to prevent evaporation, which were discussed earlier in the
sections 4.2.3 and 4.2.4.
In order to test the liquid lid concept in practice, a tryptic digest of myoglobin was
performed in a 15 nL vial covered with a film of octane. Within a time frame of
90 minutes, most of the myoglobin was enzymatically converted into peptides. An
electropherogram of the peptide map is shown in Figure 17 left.
- 25 -
Erik Litborn
Figure 17: Two electropherograms showing peptide maps of myoglobin after tryptic
digests, performed in chip-based vials (15 nL).
Left: The reaction was carried out under a liquid lid of octane. Reaction conditions:
95 minutes reaction time at 37°C, myoglobin: 4 mg/mL in 4 mM NH 4HCO 3, trypsin:
0.2 mg/mL. CE conditions: 85 cm capillary length, 60 cm effective length, ID 50 µm,
OD 150 µm, 0.01 M KH 2PO4, FC134 100 µg/mL, +15 kV, 6 µA.
Right: The reaction was carried out while continuous compensation of the evaporating
water from the vial was performed. Reaction conditions: 240 minutes reaction time at
37°C, myoglobin: 8 mg/ml in 8 mM NH 4HCO 3, trypsin: 0.2 mg/mL. CE conditions: 102
cm capillary length, 72 cm effective length, ID 50 µm, OD 150 µm, 0.01 M KH 2PO4,
FC134 100 µg/mL, +15 kV, 5 µA.
It is interesting to compare this map with the results obtained from a 15 nL tryptic
digest, where the evaporation was counteracted by a continuous supply of water. The
main pattern of the peaks is very similar in both cases. However, in the
electropherogram of Figure 17 (right), some additional peaks after the last three large
peaks are seen. At present, we are not certain about the origin of these peaks. We
cannot exclude the possibility that these signals are due to hydrophobic peptides.
Such peptides would be extracted or discriminated by the flow of octane. This is an
important possible drawback, which has to be considered when using the liquid lid
method.
Thus, the choice of the cover liquid is critical. Undesirable interactions and/or losses of
hydrophobic components can occur. On the other hand, there may be situations
where the removal of such components is desirable. For example, a comparative
study, where both methods, utilized in Figure 17 (left) and Figure 17 (right) are used
- 26 -
Sample Handling in Nanoscale Chemistry
in a side by side experiment, may reveal valuable chemical information of individual
components in complex mixtures.
Combining the liquid lid technology with a piezo-electric dispenser offers a unique
possibility for non-contact dosing while solvent evaporation is avoided, Figure 18.
Chip-based vial
with reaction
mixture
Piezo-electric dispenser
Drainage hole
Inlet - liquid lid
Outlet (overflow)
Piezo-electric dosing
through the liquid lid
Volatile liquid lid
Coalescence between the drop
and the reaction mixture
Figure 18: A schematic of non-contact dosing through a liquid lid using a piezo-electric
dispenser. Dispensing is possible to perform without splashing of formation of satellite
droplets from the dispensed liquid.
Droplets can actually be shot through a thin solvent film and coalesce into the vial
without disintegration into smaller droplets. The droplets keep intact because the
energy needed to overcome the surface tension for creating new smaller droplets is
higher than the kinetic energy within the droplet leaving the dispenser. In this way, a
non-contact dispensing of very small volumes of sample can be performed without
problems of matrix evaporation. In fact, a “closed system” is obtained but the
flexibility of an “open system” is maintained. However, it is very important that the
thickness of the lid is thin (ca 0.5 mm) and the dispensing parameters are optimized. In
addition, parameters such as viscosity and density of the dispensed solution as well as
the solvent lid will affect the success of the operation.
- 27 -
Erik Litborn
4.3 Influence of increased surface/volume ratio (SVR)
An important parameter that is affected by miniaturization of the reaction volume is
the SVR, Figure 19. When the volume is reduced below 0.5 µL, the SVR increases
rapidly.
Surface to volume ratio (m -1 )
40000
30000
Cylinder
Cone
Sphere
Nanovial
20000
10000
0
0
0,1
0,2
0,3
Volume (µL)
Figure 19: A plot showing the change of SVR when the volume is reduced for different
geometric structures compared with a chip-based nanovial (according to Figure 1). The
size of the cylinder and cone are determined using the length (l), height (h) and diameter
(d); cylinder l=4d, cone h=4d. The ratio increases rapidly for all structures when the
volume is reduced to ca 0.05 µL or less!
In the experiments with the open nanovials where tryptic digests of myoglobin were
performed, an increased reaction performance was observed for reactions carried out
in nanovials compared with plastic vials of conventional size (Paper III). It is
reasonable to assume that these observed differences are related to the different
surface properties and the SVR since it is well known that surfaces can adsorb
proteins or peptides [104, 156-161]. During surface adsorption, native myoglobin
molecules mainly form a random coil structure (a type of a surface-induced denaturing
state of the myoglobin molecules) [157, 161], which is likely to result in an increased
accessibility of the enzyme to the protein. For a continued digestion process via
surface-adsorbed myoglobin, the digestion products have to be exchanged with new
myoglobin at the vial surface. This is likely to occur since it has been shown in terms
of surface binding energy, that proteins are preferentially adsorbed compared to
peptides [162]. In addition, Vroman has suggested that a consecutive replacement of
adsorbed proteins to more surface-active ones occurs, even though the latter are
present in lower concentrations in the solution [163, 164]. Other factors such as the
shorter diffusion length in the nanovials could also enhance the digest due to faster
mass transfer-related association of protein molecules with the vial surfaces.
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Sample Handling in Nanoscale Chemistry
In order to investigate the influence of the difference in SVR, an additional experiment
was performed. Two identical digests in 100 µL volumes, using conventional plastic
vials, were carried out during 6 hours. In order to simulate the reaction conditions in a
nanovial, a thin gold-wire (l = 5 m, d = 100 µm) was bundled and inserted into one of
the vials, which roughly resulted in the same surface-to-volume ratio as in the case
where the digest was performed in a nanovial (nanovial: ~15000 m -1, conventional vial:
~900 m-1). The experiment showed that significantly more peptide material was
produced in the vial with the gold-wire, Figure 20.
myo
myo
A
B
Figure 20: Two electropherograms showing peptide maps of myoglobin after tryptic
digests, performed in a conventional plastic vial (right SVR ~900 m-1) and with an inserted
gold wire (length 5 m, diameter 100 µm) to simulate the conditions in a nanovial (left SVR
~15000 m-1). The conversion from protein to peptides can roughly be calculated from the
ratio between the peak height of a representative peptide clearly notable in both
electropherograms (A and B) and the peak height of the remaining undigested myoglobin
(myo). The yield improvement of peptide was a factor 3.5 (expressed as relation between
peak height ratios), which was consistent with the results achieved from the earlier
experiments, using nanovials and conventional vials, showing a corresponding peak height
relation of 3.3.
Reaction conditions: 100 µL reaction volume, 8 mg myoglobin/mL, 0.2 mg trypsin/mL,
8 mM NH 4HCO 3 pH 7.9, 6 hrs 37°C.
CE conditions (Beckman P/ACE system 5500): separation capillary ID 50 µm, OD 375 µm,
total length 57 cm (50 cm to detector), 10 mM phosphate buffer pH 7, 100 µg FC134/mL,
+30 kV 9.5 µA, 5 s pressure injection.
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Erik Litborn
In this typical case, an increased surface is beneficial for the reaction. In other cases,
surface interaction can be detrimental and surface treatment is very important for a
successful reaction e.g. for PCR [165, 166]. However, in our work with miniaturization
of the PCR using a liquid lid of octane (Paper VI), the sample formed a spherical
droplet at the bottom of the octane phase due to the hydrophobic character of the
surface. Consequently, surface contact with the vial is minimal. Using octane as a
barrier against evaporation of water, amplification of DNA was performed in 50 nL
PCR volumes. We noticed that the polymerase concentration needed to be increased
when the reaction volume was reduced. This might be a result of an enrichment of
polymerase molecules at the water/octane interface due to hydrophobic interaction.
This would decrease the number of polymerase molecules free to participate in the
amplification. It could also be that the presence of octane decreases the polymerase
activity.
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Sample Handling in Nanoscale Chemistry
5. Analysis of nL-pL sized volumes of sample
The analytical methods described in this chapter are limited to the techniques used in
the thesis.
5.1 Capillary electrophoresis
5.1.1 Principle
Electrophoresis was introduced by Tiselius [167], and is a separation technique well
suited for the analysis of biomolecules in a miniaturized system. CE was first
demonstrated by Hjertén [168, 169], and was further developed by Everaerts et al.
[170] and Jorgenson et al. [171-173]. Today, CE is an established separation tool and
several commercial machines are available. This has resulted in many published
articles with applications such as separations of proteins, peptides, DNA, nucleotides,
amino acids, small inorganic ions, whole cells, particles, and several review articles
have been published [174-177]. Books describing the theory of the technique are also
available [178, 179].
The separation of analytes in free solution using electrophoresis is based on
differences in mobility for charged species in an electric field. The ion velocity, v, is
given by equation (7)
v = µe ⋅ E
(7)
where µe is the electrophoretic mobility and E is the electric field. Using the ion charge,
q, the viscosity of the separation media, η, and the radius of the solute, r, the
electrophoretic mobility can be calculated according to equation (8).
µe =
q
6πη r
(8)
Sample injection into the separation capillary can be performed electrokinetically or
hydrodynamically using only a few nanoliter of the sample. The conductivity of the
sample will greatly affect the quality of the electrokinetic injection. When the sample
conductivity is lower than that of the separation buffer, the analytes will be injected in
a narrow band, which is necessary for good separation efficiency. However, during the
electrokinetic injection, a discrimination of the analytes occurs which favors analytes
with high electrokinetic mobility. This discrimination can be avoided by performing
the injection hydrodynamically but then the band broadening will increase due to
Taylor dispersion.
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Erik Litborn
Most detection principles for CE have been developed from HPLC detectors [180].
The most common detector for CE is on-column UV-VIS absorbance. The detector is
simple to use but due to the small path length within the capillaries, the concentration
sensitivity is moderate (ca 10-6 M). A more sensitive detection principle is
fluorescence; especially laser induced fluorescence (LIF), which offers sensitivity on
the order of ca 10-12 M [181]. Other detection methods based on electrochemical
principles can be used e.g. amperometric, potentiometric and conductivity detection.
CE has also been interfaced on-line with electrospray mass spectrometry [182].
5.1.2 EOF
Normally, capillaries made of fused silica, with inner diameters in the range
50-100 µm, are used as separation channels. Due to the negatively charged silanol
groups (SiO - at pH>ca 2-3) on the inner surface of the capillary, positively charged
buffer ions in the separation media are attracted and enriched close to the surface. A
double layer of ions is formed where the inner stagnant layer is bound to the wall and
the outer layer is more diffuse. When a voltage is applied longitudinally over the
capillary, the outer diffuse layer moves against the cathode. A bulk flow of the
separation media also starts to move against the cathode due to the friction (viscosity)
within the fluid. This bulk flow is termed electroosmotic flow (EOF). The flow profile
is essentially flat and no additional band broadening is induced due to the bulk flow.
For this reason, much higher separation efficiencies can be achieved compared to a
pressure driven system with a parabolic flow profile.
There are several other techniques developed from CE or CZE (capillary zone
electrophoresis). In micellar electrokinetic capillary chromatography (MECC),
surfactant added to the separation buffer creates micelles, which will interact with
hydrophobic solutes. Depending on the surfactant used, the micelles will move either
towards or with the EOF and thus affect the separation of solutes. DNA fragments are
not possible to separate in a CZE system since DNA fragments have the same mass to
charge ratio. Therefore, a sieving media or a gel must be created within the capillary
[183] - capillary gel electrophoresis (CGE). The sieving effect obtained by the gel
results in shorter migration times for shorter DNA fragments. In capillary isoelectric
focusing (CIEF), ampholytes with a wide range of pI are used to create a pH gradient.
During separation, solutes will migrate through this gradient until the pH is equal to
their pI. Capillary isotachophoresis (CITP) is a technique commonly used for
concentration of the solutes. The separation is performed using a buffer system
consisting of a leading buffer with a higher mobility in front of the solutes and a
terminating buffer with lower mobility after the solutes. When the separation voltage is
applied, the solutes will arrange themselves in consecutive zones behind the fastest
moving leading ion. The recent technique is CEC where CE separations are performed
within capillaries filled with chromatographic media [184]. In this hybrid between
HPLC and CE, the plug-flow of electroendoosmosis is utilized to increase the
separation efficiency.
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Sample Handling in Nanoscale Chemistry
5.1.3 Wall adsorption
The strong negatively charge on an untreated fused silica capillary wall, due to the
silanol groups, often attracts proteins during analysis with CE. This can be observed as
peaks which become broad or even missing peaks in an electropherogram obtained
after separation of proteins. The adsorption can be decreased by a number of methods,
such as raising the pH above the pI of the protein (electrostatic repulsion between the
protein and the wall) or lowering the pH in order to protonate the silanol groups. Also,
an increase of the buffer strength promotes competition between cationic buffer ions
and positively charged sites of the protein during interaction with the negatively
charged capillary wall. Different buffer additives and modifications of the capillary
wall have also been proposed.
A method developed in our laboratory has showed to be very useful to decrease
protein adsorption in CE [185-187]. The method is based on the addition of a
fluorinated cationic surfactant (FC134) to the separation buffer. The positively charged
head group of the surfactant will be adsorbed to the capillary wall due to electrostatic
attraction and a first layer is formed. Due to the strong hydrophobic forces between
the tails of the surfactant molecules, now bonded perpendicular to the wall, a second
layer of surfactants is created of the non-bound surfactant molecules. The bilayer
formed will cause a reversal of the EOF due to the positive charge characteristic of this
layer. Consequently, the CE analysis should be performed in a reversed potential
mode.
5.2 Matrix Assisted Laser Desorption/Ionization Time of Flight Mass
Spectrometry
5.2.1 Principle
In 1987-1988, MALDI-TOF MS was introduced by Karas et al. [188, 189] and in 1988
it was reported for the first time to use an UV-laser for desorption of bioorganic
compounds with molecular weights above 10 kDa [190].
Prior to analysis, the analyte is mixed with a matrix in a solution using a molar ratio
ranging from 1:100 to 1:50000. Several different matrices can be used depending on
the type of analyte [111] e.g. for proteins 2,5-dihydroxy benzoic acid or succinic acid
are commonly used. The matrix fills several important purposes. First, it dilutes the
analyte to prevent association of analyte molecules. Second, it absorbs energy and
transfers it gently to the analyte, and thereby protects against analyte decomposition.
Finally, it probably enhances ion formation of the analyte by photoexcitation or
photoionization of the matrix followed by proton transfer to the analyte molecule
[191].
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Erik Litborn
Analyte ions are formed by shooting a short laser pulse against a dried spot of the
matrix/analyte mixture. After acceleration of the ions to a fixed kinetic energy by an
electric field, the masses of the ions are analyzed in a TOF spectrometer. After
determination of the velocity of the ions by measuring the time to pass the flight tube,
the mass to charge ratio (m/z) can be calculated. The time frame for an entire analysis
after the sample preparation is less than 5 s per sample spot, which opens the
possibilities for high throughput analysis.
5.2.2 Sample preparation (Paper I)
The quality of a mass spectrum is very dependent on the sample preparation [192,
193]. A commonly used method is the dried droplet method, which includes
premixing of the analyte and the matrix solution and subsequently deposition on a
metal target where the spot is left to crystallize. Unfortunately, the quality of the
crystals formed within the spot is not uniform which leads to poor reproducibility
within the same spot and between different spots. In many cases, hot areas within a
sample spot must be found by shooting the laser at several different positions. Several
different sample preparation methods have been proposed to increase the
homogeneity and quality of the matrix/analyte crystals e.g. fast evaporation, crystal
crushing, spin coated drying, spray methods, recrystallization and seed layer
preparation. The goal for all these methods is to reduce the size of the crystals in order
to achieve more homogenous composition and thus a better reproducibility. A
complement to the listed methods is to prepare a sample spot with a size
corresponding with the laser spot. This has been performed using chip-based picoliter
vials with an individual volume of 250 pL (Paper I). Further, the fast evaporation of
sample solvent, due to the small volume of sample used, also resulted in the formation
of very small matrix crystals which is beneficial for good shot to shot reproducibility.
Using the 250 pL vials, single-shot mass spectrum of as low amounts as 2.5 amol
bradykinin and 25 amol of horse-heart cytochrome c were obtained, see Figure 21.
- 34 -
Sample Handling in Nanoscale Chemistry
Figure 21: Single-shot MALDI spectrum obtained from the 250 pL vials containing
(a) 2.5 amol of bradykinin and (b) 25 amol of horse-heart cytochrome c.
Recently, targets with hydrophobic surfaces equipped with hydrophilic anchors have
been presented 7. The use of such targets results in focused matrix crystals with high
concentration of the analyte, which lowers the limit of detection. Also, a new MALDI
target modification has been presented to enable the use of liquid matrices in
commercial ion sources [194]. This is beneficial for IR-MALDI since the use of solid
matrices together with IR ionization leads to increased analyte depletion in the
irradiated sample spot. When liquid matrices are used, the irradiated spot can replenish
itself by diffusion from the bulk sample solution. This is beneficial in order to increase
the homogeneity of the analyte/matrix mixture during collection of several mass
spectra without reposition of the target.
5.3 Fluorescence measurements
Fluorescence and luminescence detections within miniaturized systems are very
beneficial due to the high sensibility and the possibilities to perform measurements in
a non-contact mode. One of the most useful features is the possibility to perform
parallel measurements in platform [5] and array systems [52, 195-197] using CCD
cameras [198] or photon multiplier tube (PMT) detectors.
We have used LIF detection to follow the titration of small (nL) volumes using piezopipettes for an incremental addition of reagent (Paper II). In initial experiments,
7
AnchorChip, Bruker Daltonics, Germany
- 35 -
Erik Litborn
methyl-red was used as an acid-base indicator and the progress of the titration was
followed by direct monitoring, using a CCD-camera, without any particular optics or
illumination arrangements. However, the detection system based on light absorption
demanded very high indicator concentration in order to obtain an appreciable
signal/noise ratio. This led to large errors, since both the sample and the indicator were
titrated. Therefore, a fluorescence detection procedure, using fluorescein as an acidbase indicator was adopted. In this way, a very low concentration (10 µM) of
indicator could be employed. The suitability of this approach has earlier been
demonstrated by Wolfbeis et al. [138]. These authors used a blue light emitting diode
and a bifurcated optical fiber. Although attempts were made to miniaturize the titration
cell, their experiments were limited to sample volumes of 500 µL, determined by the
size of the optical fiber and the precision of the burette.
The development of intercalating dyes for determination of double stranded DNA has
resulted in new sensitive quantification reagents e.g. PicoGreen ® [199, 200]. A
modified form of the protocol was used to perform a fast determination of successful
amplifications in our work with the miniaturized PCR using a liquid lid (Paper VI).
The PicoGreen ® molecules inhibit the polymerase so the dye must be added after the
thermocycling in a separate step prior to the analysis. Also, all DNA molecules present
in the sample will be detected, even amplified non-specific DNA and primer-dimer.
Therefore, a CGE analysis of the DNA samples should be performed as well in order
to confirm the results.
The amplification of DNA can also be followed in real-time by using a fluorogenic
target-specific probe (TaqMan ®) [166, 201, 202]. The probe is an oligonucleotide with
both a reporter and a quencher dye attached. The probe anneals between the forward
and reverse primers if the target sequence is present. During PCR thermocycling,
annealed probes are cleaved by the 5’ nuclease activity of the polymerase and the
reporter dye is separated from the quencher dye. This separation results in a detectable
signal from the reporter dye. Additional reporter dye molecules are cleaved from their
respective probes within each cycle, resulting in a proportional increase in
fluorescence intensity. When working with miniaturized systems, the approach of
using target-specific probes is preferable since all reagents can be dispensed prior to
the thermocycling.
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Sample Handling in Nanoscale Chemistry
6. Future outlooks
Nanoscale chemistry can be performed in different kinds of miniaturized reactors.
Two different modes are possible: continuous flow reactions or batch-wise operations.
Each mode has its own usefulness, advantages and limitations. The final choice of
reactor type has to be based on different application criteria, e.g. how reagents can be
added (liquids as well as solids), to which extent cross-contamination must be
eliminated, and if a removal of by-products is necessary. Problems with solvent
evaporation can be critical as discussed earlier. Today’s chemical laboratories are
highly automated in order to meet the demands of high-throughput capacity. For
example, in the next generation platforms for screening of drug candidates, it is likely
that as many as 1,000,000 samples/day will be processed. For simple assays, the
capacity available today reportedly is already 245,000 samples/day 8. The amount of
sample and reagents needed to perform all these reactions would be enormous without
miniaturized systems. Reactions must be performed in parallel to obtain the highthroughput capacity. From this point of view, reactions performed in a batch-wise
mode could be preferable compared to continuous flow systems. However, flow
systems could also be attractive if a segmented flow technology is utilized with e.g.
1,000 reaction tubes and 1,000 separate fluid segments in each reactor. However, it will
not be easy to deal with cross contamination problems. Flow reactors in a format of a
CD are already under commercial development. The format enables the use of
centrifugal forces to distribute samples and reagents in parallel channel systems.
However, even though the parallel capacity within each CD is e.g. 384 reactors, 2,600
CDs would be needed every day to reach the throughput capacity of 1,000,000
samples/day. Miniaturized vials for batch-wise reactions arranged in a high-density
format (1,000*1,000 reactors) could therefore be more competitive. The standard
format for biochemical assays in today’s laboratories is the 96-well platform.
Operations like solid-phase extraction [203], liquid-liquid extraction [204, 205], and
filtration can be performed within this format in a highly automated way. Addition of
reagents and samples is performed with multiple dispensers arranged in the same
format. In a near future, 384- and 1536-well formats will probably replace the 96-well
platform. The “high-density-record” demonstrated so far is the 9600-well format [8]
and it is just a question of time when platforms with increased density will be utilized.
The degree of miniaturization, which is realistic to utilize in practice is dependent on
the amount of reaction product needed. In applications where only an analytical
yes/no answer is required, miniaturization can theoretically be pursued until the
ultimate detection limit is reached (i.e. single molecule detection). However, in a
screening application where the reaction product needs to be used in further studies,
miniaturization of the reaction step is feasible only if subsequent steps also can be
performed in a compatible miniaturized format. Today, this is not the case. In the
future, the primary screening stage as well as lead optimisation and evaluation should
all be performed in miniaturized systems to take full advantage of all the benefits a
miniaturization offers.
8
CyBiTM-Screen-Machine, http://www.cybio-ag.com
- 37 -
Erik Litborn
Many of the dispensing tools suitable for automation e.g. piezo-electric dispensers,
can deal with very small volumes (down to pL). However, rather large volumes of
reagents are needed to fill such dispensers. The development of a new miniaturized
inkjet-type device useful for dispensing picoliter samples from volumes less than 1 µL
is highly desirable. The dispensers available today are useful for parallel dispensing of
only a few rows of sample at a time. This will probably not fulfill the future
requirements for high-throughput sample and reagent delivery. Therefore, new
dispensers need to be developed to suit the miniaturized reactors in the future highdensity platforms. Further, the development of cheap disposable piezo-electric
dispensers that could be utilized for sampling of blood, body fluids etc., would be
another milestone in miniaturized chemistry. However, a major issue of sampling will
always be the logistic problem, where different samples will demand individual storage
containers in order to avoid cross-contamination.
The need for parallel analysis is increasing dramatically also in other areas, e.g. in the
rapidly increasing research area of proteomics where MALDI-MS enables protein
identification in an unprecedented way [206]. However, protein separation of low
abundant proteins often results in non-visible protein spots in the 2D gel, which is
employed to separate the proteins. The spots can be analyzed by a scanning procedure
using MALDI-MS [207]. After extraction of the digested proteins from the 2D gel
(size 16*16 cm2) 36 days of continuous measurements would be required to collect all
mass spectra. When the huge amount of data has been collected, the evaluation of the
results will still require a substantial time of work. Proteomics is a typical example,
where the reactions should be performed following a more clever strategy in order to
reduce the number of samples to evaluate, e.g. by using a first sorting procedure of
relevant samples, e.g. based on a fast fluorescence measurement.
Recently, a break-through concerning LIF detection for DNA separation using parallel
capillary gel electrophoresis has been presented [197]. The capillaries are rearranged
from a two-dimensional array configuration into a three-dimensional capillary bundle.
The fluorescence light from the labeled DNA fragments, at the position for laser
excitation, is guided via total internal reflection to the end of the capillaries and imaged
onto a CCD camera. With this detector, parallel CGE analysis of PCR products
amplified in a high-density open well platform should be possible, offering a higher
throughput and separation efficiency compared to current chip-based CGE devices.
In summary, this thesis has described a number of methods for performing nanoscale
chemistry. These methods should provide some useful alternatives and/or new inputs
to methods presently being used in the research area of chip-based chemistry. The
trend today, in my opinion, is that many researchers in the field often give higher
priority to technical developments as such than to obtaining performance for realworld applications. My hope is that the presented results (Paper I-VI) will give
inspiration to develop new ideas to use simple devices in a more creative way.
- 38 -
Sample Handling in Nanoscale Chemistry
DENSO Research Laboratories, Japan
- 39 -
Erik Litborn
- 40 -
Sample Handling in Nanoscale Chemistry
7. Acknowledgements
First, I want to thank my supervisor, Prof Johan Roeraade, for supporting this work,
for his superior enthusiasm for research, his never-ending efforts in improving my
English writing and his never-ending stream of ideas.
I also would like to thank:
• Dr Åsa Emmer for introducing me into the practical use of capillary
electrophoresis, for our common research projects and your comments on the
manuscript of this thesis. You will always remind me that parents are the ones
that really have high capacity in performing work in parallel
• Dr Mårten Stjernström for your knowledge within LIF detection and
mathematics, especially in our work with the piezo-electric nanotitrations
• my other two room-mates Dr Peter Lindberg and Johan Sjödahl for being nice
colleagues always willing to give ideas for solving problems
• Dr Anders Hanning for your hard and straight way to give your opinion about
sometimes-crazy ideas popping out of my head - I appreciate it! But I will
never forgive you for abandoning OPEL…..
• Matthew Rice for checking my English in this thesis and his helping hand with
photography, computers and printers.
• to Ola Berntsson I can only say: “Watch your back, Tyrrnanosaurius is always
looking for you!!” Thank you for your “kind” generosity in delivering frags!
• all other present and former colleges at the Department of Chemistry,
Analytical Chemistry, KTH
• Dr Anders Olsson for the fabrication of micromachined devices
• Jens Rytte for taking part in the work with the miniaturized PCR
• my parents Bertil and Isabella; and my sisters Karin and Ingrid with families
for always supporting me
• and finally my lovely Ildze for always being around and helping me with
everything: PoK/E!
The Swedish Natural Science Research Council (NFR), the Swedish Research Council
for Engineering Sciences (TFR) and the Swedish Foundation for Strategic Research
(SSF) are gratefully acknowledged for financial support.
- 41 -
Erik Litborn
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Sample Handling in Nanoscale Chemistry
8. References
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[6]
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FK. Nelson, H. Iwasaki, K. Hager, M. Gerstein, P. Miller, GS. Roeder, M.
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LJ. Kricka Miniaturization of Analytical Systems Clin. Chem. 1998, 44, 2008-2014.
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[14]
J. Drott, L. Rosengren, K. Lindström, T. Laurell Porous Silicon Carrier Matrices
in Micro Enzyme Reactors - Influence of Matrix Depth Mikrochim. Acta 1999, 131,
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W. Ehrfeld, V. Hessel, H. Lehr In Microsystem Technology in Chemistry and Life
Science; Manz, A., Becker, H., Eds.; Springer-Verlag: Berlin Heidelberg, 1998; Vol.
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