28). Interestingly, a recent theoretical study
on the HF dissociation-recombination reac-
tion (29) has led to a conclusion in favor of a
three-stage proton-transfer reaction mecha-
nism and has found a concerted reaction
mechanism upon diffusional encounter to be
energetically unfavored.
References and Notes
1. P. L. Geissler, C. Dellago, D. Chandler, J. Hutter, M.
Parrinello, Science 291, 2121 (2001).
2. D. Marx, M. E. Tuckerman, J. Hutter, M. Parrinello,
Nature 397, 601 (1999).
3. R. P. Bell, The Proton in Chemistry (Chapman and Hall,
London, ed. 2, 1973).
4. D. N. Silverman, Biochim. Biophys. Acta 1458, 88 (2000).
5. W. Ku¨hlbrandt, Nature 406, 569 (2000), and refer-
ences therein.
6. M. Eigen, W. Kruse, L. De Maeyer, Progr. React. Kinet.
2, 285 (1964).
7. M. Eigen, Angew. Chem. Int. Ed. 3, 1 (1964).
8. A. Weller, Progr. React. Kinet. 1, 187 (1961).
9. J. L. Skinner, H. P. Trommsdorff, J. Chem. Phys. 89,
897 (1988).
10. A. J. Leggett et al., Rev. Mod. Phys. 59, 1 (1987).
11. P. Ha¨nggi, P. Talkner, M. Borkovec, Rev. Mod. Phys.
62, 251 (1990).
12. A. Oppenla¨nder, C. Rambaud, H. P. Trommsdorff, J.-C.
Vial, Phys. Rev. Lett. 63, 1432 (1989).
13. K. Ando, J. T. Hynes, Adv. Chem. Phys. 110, 381 (1999).
14. T. Elsaesser, in Ultrafast Hydrogen Bonding and Pro-
ton Transfer Processes in the Condensed Phase,T.
Elsaesser, H. J. Bakker, Eds. (Kluwer, Dordrecht, Neth-
erlands, 2002), pp. 119–153.
15. L. M. Tolbert, K. M. Solntsev, Acc. Chem. Res. 35, 1 (2002).
16. E. Pines, D. Pines, in Ultrafast Hydrogen Bonding and
Proton Transfer Processes in the Condensed Phase,T.
Elsaesser, H. J. Bakker, Eds. (Kluwer, Dordrecht, Neth-
erlands, 2002), pp. 155–184.
17. A. Douhal, S. K. Kim, A. H. Zewail, Nature 378, 260 (1995).
18. Practical experimental issues concern limited time
resolution in time-correlated single-photon count-
ing and excited-state absorption in pump-probe
spectroscopy.
19. E. Pines, D. Huppert, N. Agmon, J. Chem. Phys. 88,
5620 (1988).
20. E. Pines, B.-Z. Magnes, M. J. Lang, G. R. Fleming,
Chem. Phys. Lett. 281, 413 (1997).
21. L. T. Genosar, B. Cohen, D. Huppert, J. Phys. Chem. A
104, 6689 (2000).
22. T.-H. Tran-Thi, T. Gustavsson, C. Prayer, S. Pommeret,
J. T. Hynes, Chem. Phys. Lett. 329, 421 (2000).
23. Bands between 950 and 1250 cm

1
are associated
with motions of the SO
3
–
groups and do not show
large changes upon proton transfer of HPTS.
24. Details of the experimental methods and modeling
can be found at Science Online.
25. The 1539 cm

1
band of the photoacid and the
photobase band at 1503 cm

1
cannot be detected in
the case of high acetate concentrations because of a
nearby acetate band.
26. This same value is obtained by analyzing solvatochro-
micshifts of the electronicabsorption bands of HPTS
as function of acetate concentration.
27. A. Szabo, J. Phys. Chem. 93, 6929 (1989).
28. W. C. Natzle, C. B. Moore, J. Phys. Chem. 89, 2605 (1985).
29. K. Ando, J. T. Hynes, J. Phys. Chem. A 103, 10398 (1999).
30. G. Zundel, Adv. Chem. Phys. 111, 1 (2000).
31. The progress has benefited from the financial support
of the Deutsche Forschungsgemeinschaft (Project
DFG NI 492/2-2) and the German-Israeli Foundation
for Scientific Research and Development (Project GIF
722/01) and from comments by C. Lienau and H.
Fidder; B.Z.M. acknowledges financial travel support
through the LIMANS Cluster of Large Scale Laser
Facilities (Project MBI000228).
Supporting Online Material
www.sciencemag.org/cgi/content/full/301/5631/349/DC1
Materials and Methods
SOM Text
16 April 2003; accepted 4 June 2003
Programmed Adsorption and
Release of Proteins in a
Microfluidic Device
Dale L. Huber, Ronald P. Manginell, Michael A. Samara,
Byung-Il Kim, Bruce C. Bunker*
A microfluidic device has been developed that can adsorb proteins from solution,
hold them with negligible denaturation, and release them on command. The active
element in the device is a 4-nanometer-thick polymer film that can be thermally
switched between an antifouling hydrophilic state and a protein-adsorbing
state that is more hydrophobic. This active polymer has been integrated into
a microfluidic hot plate that can be programmed to adsorb and desorb
protein monolayers in less than 1 second. The rapid response characteristics
of the device can be manipulated for proteomic functions, including preconcentra-
tion and separation of soluble proteins on an integrated fluidics chip.
Microfluidic systems are being developed that
can separate, purify, analyze, and deliver bi-
omolecules (1–3), but, as system dimensions
become smaller, interfacial interactions begin to
dominate device performance. For example, ad-
sorption of proteins onto surfaces can result in
fouling, the denaturing of the proteins, and
consumption of precious samples. Interfacial
interactions are often manipulated with self-
assembled monolayers (SAMs). Although
globular proteins tend to adsorb on hydrocar-
bon-terminated SAMs, termination with
polyethylene oxide (PEO) results in an anti-
fouling surface (4–6).
Passive SAMs are widely used in microflu-
idics, but it is becoming clear (7, 8) that greater
functionality can be provided in more sophisti-
cated separations and sensor systems with the
use of active coatings that can change their sur-
face chemistry in response to on-chip stimuli
such as applied voltages, heat, or light. With
active coatings, the surface can be programmed
to adsorb or release proteins for applications
such as protein preconcentrators for on-chip two-
dimensional protein separations. We describe a
device in which reversible protein adsorption has
been demonstrated with the use of thermal acti-
vation of a polymeric film.
The active material in this switchable protein
trap is an end-tethered monolayer of poly(N-
isopropylacrylamide) (PNIPAM). This polymer
exhibits a lower critical solution temperature
(LCST), a temperature above which the poly-
mer becomes insoluble, in water at about 35°C
(9–11). At room temperature, the polymer
swells in water to create a relatively hydrophilic
surface with a water contact angle that can be as
low as 30°. Above the transition temperature,
the water is expelled, the polymer collapses, and
the surface becomes less hydrophilic, with a
water contact angle that can approach 90° (Fig.
1A). PNIPAM-functionalized particles and bulk
hydrogels interact more strongly with proteins
above the LCST (12, 13). At room temperature,
the adsorption of large globular proteins such as
human serum albumin (HSA) is negligible on a
tethered PNIPAM film (Fig. 1B) and is compa-
rable to that on PEO SAMs. Above the LCST,
HSA adsorption is extensive. Complete protein
monolayers form at rates comparable to those
seen on hydrocarbon-terminated octadecyltri-
chlorosilane (ODTS) surfaces. For large globu-
lar proteins such as HSA, complete desorption is
normally observed on cooling the PNIPAM
films to room temperature.
In order for a film to be usable in a reversible
protein trap for microfluidics, at least three re-
quirements must be met: (i) the polymer needs
to be in a configuration that supports the desired
thermally activated phase transition, (ii) films
must be robust and strongly attached to the
surface, and (iii) protein adsorption must be
reversible and rapid. The first two requirements
are met by synthesis routes involving chain-
grafting (10) or the in situ free radical polymer-
ization of NIPAM on functionalized SAMs (14,
15). For in situ polymerization, the SAM chains
are covalently bound to oxide-terminated sur-
faces with the use of silane coupling agents,
satisfying the need for strong, robust surface
attachment. The SAM chains are terminated
with functionalities that generate free radicals at
chain ends, stimulating the growth of polymer
molecules covalently linked to the SAM. Unfor-
tunately, our results indicate that, even when
such films support reversible swelling transi-
tions, reversible protein adsorption is often not
observed. For example, films we have produced
with the use of the tethered azo-radical initiator
azobis(isobutyronitrile) (AIBN) (14 ) typically
have high single-chain molecular weights
[weight-average molecular weight (M
w
) around
10
7
] and low grafting densities (on the order of
2.5  10
10
chains/cm
2
, or a chain-chain separa-
tion of 60 to 70 nm). With such films, we find
Sandia National Laboratory, Post Office Box 5800,
Albuquerque, NM 87185–1413, USA.
*To whom correspondence should be addressed. E-
mail: bcbunke@sandia.gov
R EPORTS
18 JULY 2003 VOL 301 SCIENCE www.sciencemag.org352

that small gaps open up between chains upon
collapse above the transition temperature, lead-
ing to irreversible adsorption of smaller proteins
such as myoglobin (14 ).
We have been able to eliminate this irrevers-
ible adsorption by increasing the graft density of
the polymer chains via an alternative chain-
transfer reaction on SAM chains terminated
with thiol groups. Here, free radicals are pro-
duced in solution with the use of thermal acti-
vation of AIBN and are transferred to tethered
thiols (16 ). In contrast to a conventional poly-
merization process, where chain transfer be-
tween surface-bound initiators yields a radical
and a dead initiator, chain transfer between thiol
groups exchanges a radical for a hydrogen atom,
yielding a radical and a regenerated thiol. The
result of fewer deleterious side reactions is high-
er grafting densities (10
11
to 10
13
chains/cm
2
,or
chains separated by 2 to 20 nm).
All of the tethered PNIPAM films discussed
here exhibit a swelling transition similar to that
exhibited by bulk gels. This transition, and its
impact on phenomena such as protein adsorp-
tion, can be visualized with an interfacial force
microscope (IFM) (17 ). This scanning probe
system enables us to monitor attractive and re-
pulsive interactions between functionalized
probe tips and PNIPAM-coated surfaces as a
function of separation distance in water, avoid-
ing the “snap-to-contact” problems associated
with making such measurements with the use of
an atomic force microscope (AFM). At room
temperature, PNIPAM films generate a repulsive
force on both approach and retraction of the tip
regardless of tip coating. For films with high
grafting densities (Fig. 2), this repulsion be-
comes apparent at a distance of around 100 nm
above the glass substrate. On the azo-initiated
film with aM
w
of 10
7
, the repulsion is detected
at 320 nm, which corresponds well to the
hydrodynamic diameter of single chains (18).
Above the LCST, the repulsive region collaps-
es by about a factor of 2. We believe that the
observed repulsion is because of physical con-
tact between the tip and hydrated PNIPAM
chains and that the collapse of the repulsive
force reflects the collapse of the PNIPAM film
above the LCST, consistent with reported
AFM measurements (19). When the tip is
retracted from the surface at temperatures
above the LCST, the plot has an attractive well
(the measured force drops below zero) that
reflects adhesion between the PNIPAM and
the tip. This reversible switching between re-
pulsive and adhesive states below and above
the LCST is consistent with the observed
switching in protein adsorption behavior.
To be useful in microfluidics, responsive
PNIPAM films need to be incorporated into a
device in which rapid and controlled heating and
cooling can be achieved in small volumes. Sev-
eral research groups have demonstrated that re-
sistive heater lines on silicon chips can be used to
provide localized temperature control in flu-
idic systems for applications such as the
thermally activated pumping of fluids (20).
The system we have developed for studying
PNIPAM is a micro–hot plate device (Fig.




Fig.1.(A) Water contact angle measurements obtained on an azo-initiated PNIPAM film as a
function of temperature. Cartoons indicate the corresponding state (swollen or collapsed) of the
film versus temperature. Although gels can exhibit contact angle changes as high as 60°, changes
of 20° to 25° are more common on monolayer films. (B) Ellipsometry results showing the
adsorption of HSA from a 0.5 mg/ml solution (0.05 M, pH  6 phosphate buffer) on PNIPAM-
coated surfaces relative to other model surfaces. Adsorption is negligible on PNIPAM below the
transition temperature, whereas a protein monolayer eventually forms on PNIPAM above the
transition temperature.
Fig.2.IFM results obtained in deionized
water between a hydrophobically mod-
ified glass tip (ODTS-coated) and a
tethered PNIPAM film having a high
grafting density. (A) Normal force pro-
files obtained on approach of the tip to
the surface show a repulsive force that
collapses into the surface above the
PNIPAM transition temperature (26°C
for the example shown). (B) Normal
force profiles obtained on retraction of
the tip show strong adhesive interac-
tions above the PNIPAM transition temperature (note 48°C curve).
Fig.3.(A) A photomicrograph of the hot plate showing the array of
gold heater lines on top of a 200-m-wide Si
3
N
4
membrane (central
white region). (B) Fluorescence microscopy images of fluorescein-
labeled myoglobin (green) interacting with a single heated line. (Left)
Image obtained on heating a line above the PNIPAM transition
temperature after exposure to a 0.5 mg/ml myoglobin solution
followed by a rinse in myoglobin-free buffer. (Center and Right)
Images were obtained 0.8 and 1.2 s after the hot line was turned off,
releasing a plume of protein into a stagnant solution. Stills have been
extracted from movie S1.
R EPORTS
www.sciencemag.org SCIENCE VOL 301 18 JULY 2003 353