THERMORESPONSIVE POLYMER-BASED MICRODEVICE FOR

NANO-LIQUID CHROMATOGRAPHY

Guillaume Paumier, Sovann Siv, Aur

elien Bancaud, Jan Sudor and Anne-Marie Gu

LAAS-CNRS, University of Toulouse, 7 avenue du Colonel Roche, Toulouse, France

Keywords:

PNIPAM, thermoresponsive polymer, nanoliquid chromatography, sample preparation.

Abstract:

We report here on the development of an integrated device for sample desalting and pre-concentration for

nanoLC / ESI-MS analysis combining poly-(N-isopropyl acrylamide) (PNIPAM) grafted microbeads and the

means to dynamically control their temperature. Thermoresponsive properties of PNIPAM induce switchable

hydrophobic/hydrophilic surfaces on which peptides can reversibly adsorb and desorb. The device is fabricated

on a glass or pyrex substrate with deposited Ti/Au electrodes serving as built-in resistive heating sources. Pre-

molded microﬂuidic channels and reservoirs made in PDMS are eventually assembled. Electrical and thermal

characterization together with multiphysics modeling have been performed. The SiO

surfaces of the channels

and silica beads used as carriers of the stationary phases have been end-grafted with PNIPAM and employed

to study the reversible adsorption and release kinetics of albumin-ﬂuorescein conjugates by ﬂuorescence video

microscopy. It is clearly shown albumin-ﬂuorescein complexes adsorb on beads surfaces above the transition

temperature of PNIPAM (hydrophobic state), and are released when the temperature decreases (hydrophilic

state), yet not fully reversibly.

1 INTRODUCTION

The challenge of proteomics is to develop high-

throughput and integrated approaches to identify and

understand the structure, functions and interactions of

proteins. Nano-liquid chromatography (nanoLC) in

combination with electrospray ionization mass spec-

trometry (ESI-MS) detection has become a major ex-

perimental method owing to its high separation power

and sensitivity (Ishihama, 2005). In general, pro-

teins are fractionated, isolated and digested into pep-

tides to be analysed and identiﬁed by nanoLC / ESI-

MS. Miniaturization provides a number of advantages

such as low limit of detection, small volumes of an-

alyte required and reduced intermediate manipulation

steps (Gauthier and Grimm, 2006). Thus, many ef-

forts have been made to integrate on-chip separation

devices providing the column, connection capillaries

and MS coupling via a nanospray emitter (Hern

andez-

Borges et al., 2007). Though, due to MS high sensi-

tivity to salts, peptides need to be desalted and con-

centrated on C4 or C8 columns prior to their analy-

sis (Wilm and Mann, 1996). During this step, certain

hydrophobic peptides can be lost on the hydrocarbon

surfaces because they show a greater afﬁnity to the

stationary phase as compared to the mobile one uti-

lized for desorption of the puriﬁed peptides (Peterson

et al., 2003).

We propose here a novel approach based on sta-

tionary phases prepared from poly(N-isopropyl acry-

lamide) (PNIPAM) that can reversibly adsorb and re-

lease peptides upon external activation in a purely

aqueous environment. We present the development

of an integrated device for sample desalting and pre-

concentration for nanoLC / ESI-MS analysis, com-

bining PNIPAM grafted surfaces and the means to

dynamically control their temperature by integrated

microheaters.

2 THEORY

PNIPAM is a stimuli-responsive polymer which un-

dergoes a reversible coil-to-globule transition at its

178

Paumier G., Siv S., Bancaud A., Sudor J. and Gué A. (2008).

THERMORESPONSIVE POLYMER-BASED MICRODEVICE FOR NANO-LIQUID CHROMATOGRAPHY.

In Proceedings of the First International Conference on Biomedical Electronics and Devices, pages 178-181

DOI: 10.5220/0001053101780181

 SciTePress

lower-critical solution temperature (LCST) around

◦

C. PNIPAM grafted surfaces can be switched from

a swollen, hydrophilic and non-fouling state to a col-

lapsed, hydrophobic and protein-adsorbing state us-

ing thermal actuation (Kanazawa et al., 1996; Huber

et al., 2002). Such surfaces have been previously re-

ported for spatio-temporal control of ﬂows in ﬂuidic

microsystems by our group (Sudor et al., 2006).

The idea presented here is to use PNIPAM-

decorated beads as stationary phases to trap peptides

during desalting and pre-concentration steps prior to

the nano-LC / ESI-MS analysis. The reversible tran-

sition of PNIPAM surfaces upon temperature allows

controlled adsorption and release of peptides without

the change of quality of a solvent. To increase spe-

ciﬁc surface of interaction between PNIPAM and pep-

tides, PNIPAM is grafted on micrometric silica beads

injected into the channel. The channel height is re-

duced at its center to block the beads, while its width

is widened to preserve the constant surface area.

A resistive heating device is directly integrated

on the pyrex subtrate to control the temperature in-

side the channel. Microﬂuidic pre-molded PDMS

channels and reservoirs are eventually assembled to

the substrate to form the ﬁnal ﬂuidic microsystems

(Fig. 1).

Figure 1: 3D view of the assembled prototype: pyrex sub-

strate, heating line and PDMS channel.

3 EXPERIMENTAL

3.1 Heating Device

Our heating device was made with lines fabricated

on a silicon, glass or pyrex substrate with deposited

Ti/Au (1000 / 8000

A) electrodes serving as built-

in resistive heating sources. Lines of 100 µm and

500 µm width (respectively 32.5 Ω and 6.75 Ω on

pyrex) were realized and characterized. Infrared

imaging showed the heated zone was localized around

the heater (Fig. 2). Suitable temperatures were ob-

tained for acceptable voltages: 51

◦

C for 4 V (500 µm

wide) and 7 V (100 µm wide), given that LCST

of PNIPAM is around 32

◦

C. For 500 µm-wide lines

around 4 V, we obtained a homogeneous heated zone

more than 1.5 mm wide (microﬂuidic colons are

50 µm wide). Response time in heating is very short

(< 1s); cooling happens in seconds.

Multiphysics modeling using Comsol was also

performed. First modeling results ﬁt relatively well

with experimental data (Fig. 3), however, a slight re-

ﬁnement of the model is still necessary. Work is also

underway to develop more complex heating devices

allowing more precise control of heated zones (Pau-

mier et al., 2007).

Figure 2: Infrared thermal imaging of the Ti/Au electrode

(

◦

C). The resistor appears black because of the infrared re-

ﬂection on gold.

Figure 3: Multiphysics modeling of the heating process us-

ing Comsol.

3.2 Surface Chemistry

A 500 nm SiO

layer was deposited by plasma-

enhanced chemical vapor deposition (PECVD) on

the substrate and the electrodes to provide an elec-

tric insulator, and to allow homogeneous PNIPAM

grafting. The SiO

surfaces of the channels and sil-

ica beads (used as carriers of the stationary phases)

THERMORESPONSIVE POLYMER-BASED MICRODEVICE FOR NANO-LIQUID CHROMATOGRAPHY

179

were end-grafted with PNIPAM according to litera-

ture (Hjert

en, 1985), through an intermediate silane

layer (3-trimethoxysilyl propylmethacrylate). Sur-

faces were characterized with dynamic contact an-

gle measurements and multireﬂection infra-red spec-

troscopy. The latter showed speciﬁc peaks identifying

chemical groups from PNIPAM (Fig. 4).

Figure 4: Multiple internal reﬂection infrared spectrum of

surfaces grafted with PNIPAM.

4 RESULTS AND DISCUSSIONS

4.1 Controlled Adsorption/Release of

Proteins

To prove feasibility of using beads decorated with

PNIPAM to adsorb/desorb proteins, we made our ﬁrst

experiments in capillaries. We used silica beads (5 µm

in diameter) end-tethered with PNIPAM chains in

fused silica capillaries (100/385 µm inner/outer di-

ameter). The inner surface of the capillary was end-

grafted with polyacrylamide (PAM), which is not sen-

sitive to temperature changes in the studied range.

Bovine serum albumine (BSA) - ﬂuoresceine conju-

gate (1 mg/ml) dissolved in sodium phosphate buffer

(pH 7) was injected into the capillary. When tem-

perature was increased to about 40

◦

C (above LCST),

PNIPAM chains on beads became hydrophobic; they

trapped and concentrated BSA-ﬂuorescein conjugates

on beads, making them ﬂuorescent, as shown in

Fig. 5. By decreasing the temperature below the

LCST, BSA-ﬂuorescein conjugates were released into

the solution.

The kinetics of reversible adsorption and release

was also studied. The graph on Fig. 6 shows the

adsorption and release of albumin-ﬂuorescein conju-

gates on beads. We observed the release of proteins

was not fully reversible. A proposed explanation is

the low grafting density of PNIPAM chains and con-

sequent protein adsorption on the non-modiﬁed sur-

faces of silica beads.

Figure 5: Albumine-ﬂuorescein conjugates adsorbed on 5-

µm beads (top, T > LCST ) and then released (bottom, T <

LCST ). Background ﬂuorescence is due to complexes in

solution.

Figure 6: Adsorption/release kinetics of albumin-

ﬂuorescein conjugates from glass beads functionalized

with PNIPAM (normalized units).

4.2 Beads in Microchannels

Then, we went a step further and injected silica beads

inside our PDMS microchannel. Silica beads were

not functionalized with PNIPAM at this point. The

microchannel was made of three parts. At the center,

the channel was 1250 µm wide, 1500 µm long and

4 µm high. Side channels were 50 µm wide, 3000 µm

long and 100 µm high each. This geometry allows to

trap beads where section changes.

Several sizes of beads and central height of the

middle section of the channel were tested and charac-

terized through ﬂuorescence microscopy. When these

sizes were too close, beads managed to slip inside the

central part, due to PDMS ductility: Young’s modu-

lus of PDMS depends on the mixing ratio of elastomer

and curing agent but it remains about 10

Pa (Armani

et al., 1999). We observed this phenomenon with

BIODEVICES 2008 - International Conference on Biomedical Electronics and Devices

180

5 µm beads and 4 µm central height. To prevent this,

we injected a limited amount of 10 µm beads prior

to the 5 µm beads, that were prevented from entering

the central part because of the bigger beads. Exper-

iments with PDMS devices and beads modiﬁed with

PNIPAM are currently underway.

Figure 7: Top: 3D close-up on the channel zone where

beads are blocked. Bottom: Top-view of ﬂuorescent 10 µm

and 5 µm beads blocked at the entry of the central part.

5 CONCLUSIONS

Thermoresponsive properties of PNIPAM upon tem-

perature are well known and have been demonstrated

as switchable surfaces for protein adsorption. We

demonstrated in this work the possibility to inte-

grate such switchable surfaces into ﬂuidic microsys-

tems dedicated to sample preparation for nanoLC /

ESI-MS. We have developed essential components

and know-how about heating sources, reversible pro-

tein adsorption and release, and injection of beads in

PDMS microchannels. We are now demonstrating the

feasibility of the microsystems for desalting and pre-

concentration of various peptide samples.

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