executive summary · web viewmagnetic particles for microfluidic integration have to fulfill...

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: 15 March, 2014Date of submission: 19 March, 2014Author: Assoc. Prof. Zuzana Bilkova,

Mgr. Jana Kučerova

DELIVERABLE SUMMARY SHEET

Project Number : 317742Project Acronym : LOVE-FOODTitle : LOVE Wave Fully Integrated Lab-on-Chip Platform for FOOD-Pathogen

DetectionDeliverable : D3.3Partners Contributed : UniPardubice, Institut CurieAuthors : Assoc. Prof. Zuzana BilkovaClassification : 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 Updated version submitted to coordinator

19/3/2014 1.4Modified version approved by coordinator and submitted to EC officer

FP7-ICT-2011.3.2 – Contract No 317742 – Deliverable 3.3/13

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


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.


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 super-

paramagnetic 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


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 densityDynabeads (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.


Table 2: Hydrodynamic diameter of particles and their zeta potential (measured in 0.01M phosphate buffer pH 7.2).

Diameter [nm] (DLS) Zeta potential (mV)Dynabeads MyOne Streptavidin 1421 ± 42.0 -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


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.


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-PEG30,000-

NH2 (“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 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.

Fig ure 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.


Fig ure 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


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 cone-

shaped 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.


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.


executive summary · web viewmagnetic particles for microfluidic integration have to fulfill...

Documents