Love Wave Fully Integrated Lab-on-chip Platform
for Food Pathogen Detection - LOVE-FOOD
(Contract No 317742 – Starting Date: 1 September 2012)
Deliverable 3.3
Characterized magnetic particles suitable for LOC application
Due date:
Date of submission:
Author:
15 March, 2014
19 March, 2014
Assoc. Prof. Zuzana Bilkova,
Mgr. Jana Kučerova
DELIVERABLE SUMMARY SHEET
Project Number
Project Acronym
Title
Deliverable
Partners Contributed
Authors
Classification
: 317742
: LOVE-FOOD
: LOVE Wave Fully Integrated Lab-on-Chip Platform for FOOD-Pathogen
Detection
: D3.3
: UniPardubice, Institut Curie
: Assoc. Prof. Zuzana Bilkova
: PU
DOCUMENT HISTORY
Date
Version
Description
03/03/2014
1.1
Draft
10/03/2014
1.2
1st complete version
12/03/2014
1.3
19/3/2014
1.4
Updated version submitted to
coordinator
Modified version approved by
coordinator and submitted to EC
officer
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Table of Contents
Executive Summary………………………………………………………………………………………………………………………………..…...4
Main Text……………………………………………………………………………………………...……………………………………………….…...5
1.
Introduction……………………………………………………………………………………………………………….…..5
2.
Choice of magnetic particles for microfluidic integration…………………………….…………….…..5
3.
Characterization of magnetic particles for microfluidic integration……………..…………….…..6
3.1 Zeta potential and dynamic light scattering measurement (DLS) ……………..……….…..6
3.2 Measurement of the sedimentation rate……………..…………………………………..……….…..7
3.3 Brightfield microscopy……………..……………………………………………………………………….…...7
3.4 HA or PEG coating of selected microparticles.……………..…………………………………….…..7
3.5 Evaluation of non-specific adsorption……………..………………………………………………..……9
3.6 Study of microparticles behaviour in microfluidic channel…………….………..……….…..10
3.7 Binding capacity……………..………………………………………………………………………..…..……..11
4.
Testing of particles in a microfluidic PDMS chip with fluidized bed………………………………………………..12
Conclusions……………………………………………………………………………………………………………………………..…..13
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Executive Summary
The present deliverable arose in the frame of WP3 (Sample processing and DNA extraction) - Task 3.2
(Characterization of magnetic particles for microfluidic integration) and deals with the choice of suitable
magnetic particles compatible with microfluidic systems and their characterization. This is an essential
preliminary step which had to be done before biofunctionalization of magnetic particles by specific
antibodies or nucleic acids polymers.
All magnetic particles selected for the LOVE-FOOD project were characterized by different analytical
techniques like zeta potential, dynamic light scattering, bright field microscopy, etc. After that, they were
loaded directly into microfluidic chip from PDMS recently developed by Institute Curie and their behavior
was monitored. Magnetic particles ProMag from Bangs Labs were chosen as the most promising particles
for further biofunctionalization with the aim to capture DNA from Salmonella Typhimurium.
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Main Text
1. Introduction
Realization of all tasks belonging to WP 3 was dependent on the protocol concerning the sample processing
steps; pathogen lysis, DNA extraction and DNA elution. One of two strategies how to perform all
procedures fully compatible with further amplification and detection steps is based on magnetic
microbeads as solid phase for biospecific ligands anchoring and for fixing them just inside the separation
channel or reaction chamber of microfluidic device.
Nowadays, there are a lot of commercial suppliers offering magnetic particles in the size range between 10
nm up to tens of micrometers. Although the manufacturers deeply characterize the particles they produce,
the end-customers often lack these information and have to characterize them themselves. Thorough
examination and choice of particles before their biofunctionalization with ligands of interest can prevent
subsequent problems with aggregation and undesirable particles´ behavior.
Magnetic particles for microfluidic integration have to fulfill several criterions like hydrodynamic diameter,
uniformity, good colloidal stability, quick magnetic response and high binding capacity for subsequent
immobilization of ligand of interest. The beads forming stable fluidized bed for dynamic microfluidic
capturing should exhibit a high magnetic susceptibility and magnetization to ensure high flow rate
resistance.
2. Choice of magnetic particles for microfluidic integration
The panel of microparticles differing in size, chemical properties, density and type of functional groups
were evaluated according the following criteria: large surface area, controlled (non)porous structure and
high thermal, mechanical and colloidal stability. The particles have to also be biocompatible and easily
functionalizable. We evaluated average diameter of particles, their monodispersity, type and density of
surface functional groups and level of magnetization.
Based on the results and information provided by the manufacturers on the magnetic particles shown in
Table 1, the most promising particles for microfluidic integration were selected. All of them exhibit superparamagnetic behavior allowing their easy manipulation and dispersion in the aqueous medium. Sizes
between 1 and 3 µm were preferred since it is a reasonable compromise between microparticles with
extra-large specific surface area and microparticles with high content of magnetic material, which assures
them fast magnetic response. With a view of subsequent immobilization of biotinylated oligonucleotides or
antibodies, particles covered with a layer of streptavidin were chosen. The biotin-avidin (streptavidin resp.)
complex is the strongest known non-covalent interaction between a protein and ligand. Moreover, the
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bond formation is very rapid and resistant to extreme pH, temperature or denaturing agents. Therefore,
this immobilization strategy based on molecular recognition was also selected as a validation method in
this project.
Table 1: Magnetic particles pre-selected as promising for the LOVE-FOOD project.
Type of particles and their manufacturers
size
density
Dynabeads (Life
Technologies)
Dynabeads MyOne Streptavidin C1/T1
1,05 µm
1.8 g/cm³
Dynabeads M270/M280 Streptavidin
2,8 µm
1.6 g/cm³
Chemicell
SiMAG-Streptavidin
1,0 µm
2.5 g/cm³
Micromod
micromer®-M - streptavidin
2,0 µm
1.1 g/cm³
Bangs Labs
ProMag™ 1 Series Streptavidin
0,97 µm
1.3 g/cm³
3. Characterization of magnetic particles for microfluidic integration
The quality of magnetic particles summarized in Table 1 was checked by analytical methods based on
different physical-chemical principles: zeta potential, dynamic light scattering, bright field microscopy,
measurement of the sedimentation rate and binding capacity for proteins. Dynamic light scattering is a first
choice method for size determination, zeta potential measurement for comparison of the colloidal stability;
sedimentation rate and bright field microscopy for confirmation of wholeness of the particles were applied.
The five types of magnetically active microparticles with proper characteristics were selected for next
experiments.
3.1
Zeta potential and dynamic light scattering measurement (DLS)
Zeta potential is the charge that develops at the interface between a solid surface and its liquid medium. It
is the important parameter predicating the behavior of particles in colloidal systems. Since the particles are
covered with a layer of streptavidin (protein wit isoelectric point ~ 5), their repellence is slightly reduced,
which could support the process of aggregation. Due to this reason, zeta potential has to be thoroughly
monitored in order to preserve the dispersed character of suspension of particles. Dynamic light scattering
(DLS) is a technique for measuring the hydrodynamic size of molecules and submicron particles. Data
obtained by this technique often differ from the values defined by suppliers. The need for this
measurement is thus obvious.
The results from both the above two techniques are summarized in Table 2.
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Table 2: Hydrodynamic diameter of particles and their zeta potential (measured in 0.01M phosphate buffer
pH 7.2).
Dynabeads MyOne Streptavidin
Diameter [nm] (DLS)
1421
±
42.0
Zeta potential (mV)
-19.6
±
1.34
Dynabeads M270 Streptavidin
3506
±
135.0
-16.0
±
0.87
SiMAG – streptavidin
2427
±
237.6
-8.07
±
2.53
micromer - M – streptavidin
3073
±
220.1
-36.2
±
0.07
ProMag 1 Series Streptavidin
1786
±
105.3
-20.3
±
1.48
3.2
Measurement of the sedimentation rate
Knowing that the functionalized particles have to develop homogeneous, stable but dynamic fluidized bed
just in microfluidic device also the sedimentation kinetics of magnetic particles was observed as a
parameter showing their suitability for this application. This measurement reflects the colloidal stability of
particles in time, which is the highly important aspect when choosing the proper particles for microfluidic
systems. As shown in Figure 1, the sedimentation kinetics is dependent on the size and density of particles
(larger particles support sedimentation).
Figure 1: The effect of time on sedimentation of magnetic particles for microfluidic integration.
3.3
Brightfield microscopy
The shape and wholeness of magnetic particles after the incubation with strong elution agents (urea,
glycine buffer pH 2.5) was controlled by the brightfield microscopy. All particles remained unchanged,
which supports the results from the above mentioned methods and refers to their good chemical stability.
3.4.
HA or PEG coating of selected microparticles
With the aim to minimize the rate of nonspecific adsorption during the processing of highly complex
biological material and to control the tendency of particles to self-aggregation after biofunctionalization we
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successfully modified the surface of particles by biopolymers as PEG derivatives and HA fragments. Again,
the parametrization was performed.
The hydrodynamic diameter of magnetic particles was measured using laser diffraction by particle size
analyser MasterSizer 2000. To evaluate the efficiency of coating methods as zeta potential measurement
was performed. Figure 2 documents the increased colloidal stability of HA-coated microparticles compared
with naked ones without dependence on the length of HA chains.
Figure 2: The zeta potential of naked or coated Dynabeads M270 Amine and p(GMA-MOEAA)-NH2 magnetic
particles, coated by hyaluronic acid with different Mw .
To prove the presence of HA layer on the surface of HA-coated Dynabeads M 270 Amine, particles were
analyzed by atomic force microscopy. The topography image of the well distributed as well as
agglomerated particles is illustrated on the Figure 3.
Figure 3: The topography image of the well distributeda) as well as agglomeratedb) particles measured by
AFM.
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There was detected statistically important increase in the thickness of the adsorbed water layer
determined as the length between jump-to-contact point and point of the equality of the attractive and
repulsive forces. The mean values of the water layer thickness increased from 14 nm to the 38 nm with
standard error 0.5 nm for the naked and HA-coated particles, resp. which corresponds to the high affinity of
HA to the water in comparison to the other polymers. The similar results were obtained also by laser
diffraction technique determining of their hydrodynamic diameter. All these results obviously confirmed
that all types of magnetic particles were coated with HA and the surface was modified by HA-layer with
stable linkage.
Zeta potential (ZP) and isoelectric point (pI) measurement, SEM accompanied by image analysis, IR
spectroscopy, biotin-streptavidin based interactions and anti-PEG ELISA were then applied for
parametrization of PEG-coated microparticles. Figure 4 clearly demonstrates that a presence of PEG chains
significantly affected the Kubelka–Munk infrared spectrum.
Figure 4: Kubelka–Munk Fourier transform infrared spectra of neat PGMA-COOH microspheres (denoted
“p”, solid curve), CH3-PEG30,000-NH2-PGMA microspheres (“p-PEG”, dashed curve), and CH3-PEG30,000NH2 (“PEG”, red curve). The insets a) and b) show the expanded areas of interest and band assignment.
3.5
Evaluation of non-specific adsorption
With the aim to verify the suitability of coated particles for bioaffinity assays performed even in
microfluidic layout, it was essential to evaluate the level of nonspecific sorption, tendency of particles to
agglomerate, quantify the adhesion of them to the various types of solid planar materials. Bovine serum
albumin (BSA) as the inert protein or tumor cells MCF7 have been applied for nonspecific sorption
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monitoring. The obtained results indicate significant reduction of nonspecific sorption on the surface with
HA-layer (Figure 5) or PEG-layer (Figure 5, 6), compared with naked magnetic particles.
Figure 5: The rate of nonspecific adsorption of naked or HA-coated p(GMA-MOEAA)-NH2 particles which is
proven as amount of BSA (µg) non-specifically adsorbed onto the surface of 1 mg of particles after 1 hour
incubation at RT in deionized water, the amount of BSA was determined by micro BCA protein assay kit
(Thermo Fisher Scientific, IL, USA).
Figure 6: Nonspecific adsorption of bovine serum albumin (BSA) on (1) neat PGMA-COOH, (2) CH3-PEG
30,000-NH2- PGMA, and (3) CH3-PEG2,000-NH2-PGMA microspheres.
3.6
Study of microparticles behaviour in microfluidic channel
A simple PDMS device containing seven channels was produced to evaluate the behavior of neat and
PEGylated microspheres. These highly biocompatible microparticles exhibited excellent behavior during the
contact with the material of the microchannel (PDMS, COC). These carriers were suitable for microfluidic
implementation.
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Figure 7: A sketch of PDMS chip (the inner dimensions of each channel were (w x h x l): 200 µm x 50 µm x
30 mm) used for evaluation of the adhesion of neat and PEGylated microspheres.
Minimum or no adhesion together with no aggregation of particles in the channels of the chips is a key
prerequisite for magnetic beads-based microfluidic bioassays. Two types of polymer chips – PDMS (see
Figure 7) and COC - were used to compare the behavior of microspheres before and after the PEGylation.
Substantial differences in aggregation and adhesion were recorded. Surface modification of PGMA-COOH
particles with CH3-PEG30,000-NH2 assured them higher repellence to the model protein BSA, cells and COC or
PDMS material compared to CH3-PEG2,000-NH2. Magnetic microparticles with PEG- or HA- coating can be
coupled with a ligand of interest and widely applied in microfluidics.
3.7
Binding capacity
With the aim to compare the binding efficiency of the pre-selected magnetic particles, human IgG
molecules or DNA fragments have been immobilized on the surface of the particles. The binding capacity of
particles was then compared. Data obtained for IgG are summarized in Table 3.
Table 3: Binding capacity of different commercial particles (tested with model protein – human IgG).
Particles
Binding capacity (µg of IgG / mg of particles )
Dynabeads MyOne
~ 30 µg
Dynabeads M270
~ 20 µg
SiMAG
~ 15 µg
micromer®-M
~ 20 µg
ProMag™ 1 Series
~ 80 µg
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4
Testing of particles in a microfluidic PDMS chip with fluidized bed
Together with Partner 2 and Partner 5, the behavior of selected and characterized commercial particles was
subsequently tested directly in a magnetic fluidized bed, which has been developed by Curie just for this
project. The ideal quantity of magnetic particles loaded into chip has been previously optimized by Partner
2 and has been set as 50 µg of particles. These aliquots of different particles were loaded into the coneshaped chamber of the chip. This was followed by repeated closing and opening of the fluidized bed (by a
permanent magnet) and monitoring of particle´s behavior (Figure 8). Buffers of different chemical
compositions (presence of detergents, their concentration, etc.) were tested. It was found out that larger
magnetic particles (Dynabeads M270 with the diameter of 2.8 µm) are more suitable for this microfluidic
system, since their reaction response to the attached magnet is faster and thereby the opening and closing
of the bed is more effective. PBS buffer containing Tween 20 in a final concentration of 0.05% was selected
for all further experiments with biofunctionalized particles.
Figure 8: ProMag 1 Series magnetic particles (Bangs Labs, Fishers, IN, US) in magnetic fluidized bed.
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Conclusions
All magnetic particles selected for the LOVE-FOOD project were characterized by different techniques like
zeta potential, dynamic light scattering, bright field microscopy, etc. From the list of tested particles,
magnetic ProMag Series 1 (Bangs Labs, Fishers, IN, US) were chosen as the most promising ones for the
LOVE-FOOD project. The value of their zeta potential (-20 mV) indicates their goof colloidal stability and
they excel in their large binding capacity (~80 µg of antibodies per 1 mg of particles). After their thorough
characterization they were loaded directly into microfluidic chip from PDMS recently developed by Institute
Curie and their behavior was monitored. Such particles are currently biofunctionalized with the aim to
capture DNA from model G- bacteria Salmonella Typhimurium.
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