Influence of the Debye length on the interaction of a small molecule-modified Au nanoparticle with a surface-bound bioreceptor

Article abstract of DOI:10.1039/c4cc01423e

We report that a shorter Debye length and, as a consequence, decreased colloidal stability are required for the molecular interaction of folic acid-modified Au nanoparticles (Au NPs) to occur on a surface-bound receptor, human dihydrofolate reductase (hDHFR). The interaction measured using surface plasmon resonance (SPR) sensing was optimal in a phosphate buffer at pH 6 and ionic strength exceeding 300 mM. Under these conditions, the aggregation constant of the Au NPs was approximately 10⁴ M^-1 s ^-1 and the Debye length was below 1 nm, on the same length scale as the size of the folate anion (approximately 0.8 nm). Longer Debye lengths led to poorer SPR responses, revealing a reduced affinity of the folic acid-modified Au NPs for hDHFR. While high colloidal stability of Au NPs is desired in most applications, these conditions may hinder molecular interactions due to Debye lengths exceeding the size of the ligand and thus preventing close interactions with the surface-bound molecular receptor.

Full text of DOI:10.1039/c4cc01423e

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Influence of the Debye length on the interaction
of a small molecule-modified Au nanoparticle
with a surface-bound bioreceptor†
Cite this: Chem. Commun., 2014,
0, 4947
5
Received 24th February 2014,
Accepted 30th March 2014
a
a
ab
abc
Natalia Bukar, Sandy Shuo Zhao, David M. Charbonneau, Joelle N. Pelletier
ad
and Jean-Francois Masson*
DOI: 10.1039/c4cc01423e
www.rsc.org/chemcomm
2
We report that a shorter Debye length and, as a consequence, decreased proteins, or other biomolecules) requires a very sensitive sensor
colloidal stability are required for the molecular interaction of folic acid- and is thus desirable for analytical purposes, the Au NPs must be
modified Au nanoparticles (Au NPs) to occur on a surface-bound stable in solution prior to performing the assay.
receptor, human dihydrofolate reductase (hDHFR). The interaction
The interface of NPs dictates the chemical reactivity, colloidal
10
measured using surface plasmon resonance (SPR) sensing was optimal stability, toxicity and interactions with their environment. For
in a phosphate buffer at pH 6 and ionic strength exceeding 300 mM. example, the reactivity of AuNPs in a peptide coupling reaction
Under these conditions, the aggregation constant of the Au NPs was mediated by EDC-NHS chemistry is dependent on the buffer concen-
4
À1 À1
11
approximately 10 M
s
a
and the Debye length was below 1 nm, on tration and thus the ionic strength. The pK of carboxylic acids has
the same length scale as the size of the folate anion (approximately been shown to shift by two pH units on a AuNP in comparison to the
1
2
0
.8 nm). Longer Debye lengths led to poorer SPR responses, revealing a same carboxylic acid molecule free in solution. The pK was also
a
13
reduced affinity of the folic acid-modified Au NPs for hDHFR. While high shown to shift in terms of ionic strength. Proteins form a corona on
colloidal stability of Au NPs is desired in most applications, these the surface of NPs exposed to biological media, which has a
conditions may hinder molecular interactions due to Debye lengths significant impact on biological applications of NPs including their
14,15
exceeding the size of the ligand and thus preventing close interactions toxicity.
These examples represent only a subset of notable
with the surface-bound molecular receptor.
physico-chemical properties particularly on NP surfaces.
Colloidal stability is a factor of the charge at the surface of the NP
Au NPs are commonly used in nanotechnology and nanomedicine and of the Debye length. Greater Au NP surface charge and solution
1,2
3
due to their enhanced sensing properties, as nanotemplates, due conditions favoring long Debye lengths improve the Au NP stability in
4,5
to their capacity to deliver molecules to targeted sites or in aqueous solutions. The charge is a function of the nature of the
6
therapeutic treatments. Many applications of Au NPs rely on the capping agent and of the solution pH. However, the charge becomes
interaction of functionalized Au NPs with a biological receptor or screened in solutions of high ionic strength. Thus, Au NPs bearing a
biomolecule. Several examples include tagging cells with Au NPs negative charge exhibit improved colloidal stability in solutions of
modified with a large biomolecule to interact with membrane-bound higher pH and lower ionic strength. Conversely, short Debye lengths
7
8
16
receptors, as an intracellular functional tool, or to amplify the allow close interaction of biomolecules, which facilitates biomolec-
9
response of plasmonic biosensors. Plasmonic biosensing encom- ular interactions. The Debye length of Au NPs is generally smaller than
passes a series of assays to detect molecules binding to the surface of the size of the macrobiomolecule immobilized on the Au NPs. Hence,
2
the Au NP leading to color changes or aggregation of the Au NPs.
the properties of the macrobiomolecules dictate the stability of the Au
Since the analysis of the colorimetric properties or aggregation of Au NPs. However, this assumption is no longer valid for Au NPs modified
NPs modified with a biomolecule or ligand (DNA, protein or small with small molecules and the effect of the Debye length must be
molecules) in the presence of a receptor (complementary DNA, considered when these Au NPs interact with molecular receptors.
The control of the Debye length dictates the magnitude of the
1
7
a
distance sensed by field effect transistors, of importance for DNA
D ´e partement de chimie, Universit ´e de Montreal, C.P. 6128 Succ. Centre-Ville,
Montreal, Qc, Canada H3C 3J7. E-mail: jf.masson@umontreal.ca;
Tel: +1-514-343-7342
18–21
and protein detection.
sensor sensitivity and in the stability of the receptor-target molecule.
NPs modified with biotin are commonly used to bind avidin for
Ion concentration plays an intricate role in
22
b
c
PROTEO
Centre de Chimie Verte et Catalyse (CCVC)
23
d
numerous applications. It has been reported that decreasing
the Debye length is necessary for the biotin–avidin interaction to
occur, as demonstrated to mediate aggregation of biotin-modified
Centre for self-assembled chemical structures (CSACS)
Electronic supplementary information (ESI) available: Detailed experimental
†
procedures, supplementary figures and tables. See DOI: 10.1039/c4cc01423e
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interaction of FA-modified Au NPs with the surface-bound hDHFR.
Large SPR responses indicate that FA Au NPs bind to hDHFR, thus
providing a measure of interaction under different buffer conditions.
SPR responses varied from a null signal in 10 mM Tris buffer with
154 mM NaCl to 0.62 nm for phosphate buffered saline (1Â PBS),
while the pH was kept constant at 8 (see ESI,† Table S2). A large
distribution of SPR responses was observed depending on the nature
and concentration of the buffer, and on ionic strength. General
conclusions could be extracted from the SPR experiment. Increasing
the concentration of the buffer and the ionic strength improved the
SPR response. Phosphate and Tris-based buffers performed relatively
well, while both MES buffers lead to very weak responses. hDHFR is
Scheme 1 Design of a competitive SPR biosensor for methotrexate.
Folate anion (FA)-functionalized Au NPs compete with MTX for binding
to hDHFR (colored ribbons) that is bound to a 3-MPA-LHDLHD-modified also less active in MES (see ESI,† Table S1), thus does not bind FA Au
Au surface. Low concentrations of MTX lead to a high number of binding
NPs or MTX as effectively. No further correlation could be established
events of the FA Au NPs to hDHFR and generate large SPR signals.
between hDHFR activity and SPR response for the MTX assay
2
(
see ESI,† Fig. S1 – r = 0.05). For example, hDHFR is highly active
23
vesicles with streptavidin. These considerations are also of impor- in some Tris buffers (low SPR response) and showed lower enzyme
tance in light of several recently reported assays where a competitor is activity for some phosphate buffers (large SPR response). This
immobilized on Au NPs for detection of small molecules by compe- absence of correlation reflects the difference between kinetic activity
titive assays. These assays are otherwise difficult to perform using measurements which consider initial enzyme turnover with the
plasmonic biosensors due to the small change in the refractive index substrates in a solution state, while the SPR response is related to
24
provided by this class of molecules. In some examples, the compe- binding affinity under thermodynamic conditions using surface-
titor is also a small molecule: a Au NP–stanozolol–BSA conjugate was bound enzyme. Thus, the concentration of ions and their nature
25,26
reported for the detection of stanozolol,
a Ag NP entering influence the biological binding event observed by SPR, while the
competition for chloramphenicol was developed to detect that anti- observed enzyme activity does not correlate to the SPR response.
27
biotic and our group recently developed a solution-based competi-
The SPR analysis reveals that the Au NPs interact favorably under
tive assay for the quantification of methotrexate (MTX), a common the buffer conditions known to disrupt the colloidal stability of Au
28
anti-cancer drug.
NPs. As the Au hydrosol is a thermodynamically unstable system, Au
MTX competitively inhibits human dihydrofolate reductase NPs aggregate after a certain time and lead to a change in the optical
2
9
(
hDHFR), an enzyme that is essential for cellular proliferation.
properties of NP solutions (change in the color, optical density of
34
MTX or folate competitively forms a complex with the active site of the solution, etc.). The kinetics of aggregation (coagulation) are
hDHFR. Detection is based on the competition between the free described by the equation:
MTX to be quantified, and folate anion-functionalized Au NPs (FA Au
30
35
S o o
N = N /(1 + K N t)
NPs) for binding to hDHFR (Scheme 1). A SPR sensor based on a
Cu-nitrilotriacetate-capped peptide monolayer was recently developed where N – total quantity of NPs at time t, N – initial quantity of NPs
S
o
3
1,32
to immobilize polyhistidine-tagged hDHFR onto the Au films.
The hDHFR was shown to be active at pH 8, in the following buffers: were monitored for a period of 10 days to assess the stability of the Au
-amino-2-hydroxymethyl-propane-1,3-diol (Tris), sodium phosphate NPs in each buffer. The aggregation constant could be extracted from
and K – aggregation rate constant. The UV-Vis spectra of FA Au NPs
2
and 2-(N-morpholino)ethanesulfonic acid (MES) (see ESI,† Table S1). the fraction of Au NPs still in suspension (measured from the
In addition to the influence on hDHFR, the nature, concentration intensity of the plasmon resonance at 520 nm) as a function of time
and pH of the buffer and the ionic strength of the solution play an (Fig. 1).
important role in the colloidal stability of Au NPs and may impact on
the biomolecular interactions of the FA Au NPs and hDHFR.
The highest SPR responses were obtained for a K10d of about
4
À1 À1
10 M
s . As expected, nanoparticles with very large K10d are very
The influence of the Debye length was studied for a series of unstable in solution and aggregation prevails over interactions with
buffers for which the pH was kept constant at 8 to maintain the hDHFR. The largest K10d was observed for Tris buffer with 154 mM
negative charge on the folate anion found on 13 nm spherical Au NPs NaCl, for which only 31% of the Au NPs remained in solution after
(see ESI† for detailed experimental procedures). FA Au NPs (10 nM) 10 days. In comparison, the lowest K10d was observed for buffer with
were suspended in sodium phosphate, Na-MES and Tris-Cl buffers at more than 98–99% of the Au NPs still in solution after 10 days. It is
different ionic strengths (0 to 308 mM NaCl) and buffer concentra- surprising to observe that very stable nanoparticles (low K10d) lead to
tions (10 to 100 mM). Then, the Au NP solution was mixed with nearly no response in SPR, indicative of a weak interaction of the FA
1
00 nM MTX and injected into a wavelength interrogation SPR Au NPs with hDHFR.
33
instrument. Patients undergoing MTX-based chemotherapy are
Folate anions are small molecules and must enter a relatively deep
monitored for drug clearance until the safe level of 100 nM is reached binding pocket in hDHFR for the interaction to occur (Fig. 2). The
36
in serum. Detection of 100 nM MTX was thus selected due to clinical crystal structure of folate-bound hDHFR (1DHF.PDB ) shows that the
relevance. A null response (0 nM MTX) was also measured and the pteroyl and the p-aminobenzoyl moieties are essentially shielded from
difference is reported here. A small SPR response indicates no specific the solvent inside a kinked, 16 Å-deep cavity while the carboxylate
4
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occur within the Debye length of both the Au NPs and hDHFR. The
charge of hDHFR was simulated at different pH values using the MOE
software and a titration curve was generated (see ESI,† Fig. S2). Based
on the simulated titration, a net charge (formal charge) of +4 was
calculated at pH 6, and +0.5 at pH 8. The folate anion has three pKa
a
values for the two acidic and one basic group (pK of 4.65, 6.75 and
9
.00). Thus, the folate anion bears a net negative charge at pH 8.
37
The Debye length of Au NPs can be estimated in buffers using
the equations describing the relative permittivity of NaCl solutions
1
.5 38,39
2
(
e
s
= 78.45 À d
s
C
s
+ bC
s
),
b s r b
the Bjerrum length (l = e /(4pe e k T))
and the inverse Debye length (k = 8pI I). The Debye length is obtained
from k
decrement (15.8 M ), b is a constant (3.01 M ), e is the elemental
b
À1 40
.
C is the concentration of NaCl, d is the permittivity
s
s
À1
À3/2
r b
charge, e is the relative permittivity, k is the Boltzmann constant, T is
Fig. 1 Correlation plot of aggregation constants in relation to the SPR
signal induced by 100 nM MTX in competition with FA Au NPs for surface-
bound hDHFR. Small and large aggregation constants (K10d) do not favor
interaction of the FA Au NPs with surface-bound hDHFR, as shown by the
weak SPR signal.
the temperature (293 K) and I is the ionic strength.
2
A strong correlation (r = 0.96) was observed between the SPR
response and the calculated Debye length for phosphate buffers
(Fig. 3). Other buffer conditions (Tris and MES) did not strongly
correlate with data in phosphate buffers and PBS, as MES buffers do
not favour hDHFR activity and Tris buffers destabilize folate anion-
functionalized Au NPs to a greater extent and do not allow for
interaction with hDHFR. The SPR signal in phosphate buffers
increased significantly with the shorter Debye length, suggesting that
the interaction with hDHFR is facilitated when the charge is screened
on a shorter distance. Therefore, the folate anion is capable of entering
the binding pocket of hDHFR to undergo the competitive assay for
MTX. This confirms that Debye lengths smaller than 1 nm (on the
same length scale as the size of the folate anion of ca. 0.8 nm) are
needed for optimal interaction of folic acid-modified Au nanoparticles
with a surface-bound receptor (hDHFR). This result is in agreement
with a previous observation that biotin–streptavidin interaction occurs
23
for Debye lengths on the order of magnitude of the linker length.
While it must be considered that aggregation is an improbable
cause of the larger SPR response for buffer conditions of short Debye
length. Aggregation of AuNPs on the SPR sensor would indeed lead to
a large SPR response, undistinguishable from the large SPR response
obtained under conditions of shorter Debye lengths. However, the
absence or minor contribution of aggregation is evidenced for Tris
Fig. 2 Conformation of the DHFR-bound folate anion and binding to
hDHFR. (A) The hDHFR-bound conformation of FA is according to the
crystal structure (1DHF.PDB). (A) FA: carbon atoms are in yellow, oxygen
atoms in red and nitrogen atoms in blue; the surface of hDHFR is in grey. In
the competition assay, the carboxylic acid moiety of FA (bottom part –
5
.02 Å) is bound to the Au NP, leaving the remainder of the FA molecule
exposed to the solution for binding to hDHFR. (B and C) Surface electro-
static potential mapped on hDHFR (1DHF.PDB) at pH 8 (B) and pH 6 (C);
bound FA is colored as in panel A. The color scale of electrostatics extends
from À3 (red) to +3 eV (blue) (kT/e). The pteroyl moiety (8.24 Å length) is
deeply buried, the p-aminobenzoyl moiety (7.82 Å) is partly buried and the
carboxylate (5.02 Å) is relatively exposed at the entrance of the binding
pocket, which is lined with lysine and arginine residues.
moiety lies in an indentation at the surface of the protein, albeit still
forming specific contacts with the protein (Fig. 2A). The folate anion
extends approximately 0.8 nm from the surface of the Au NPs. As the
interaction between FA and hDHFR is to occur near the surface of the
Fig. 3 Correlation plot of the Debye length in relation to the SPR signal
induced by 100 nM MTX in competition with FA Au NPs for surface-bound
hDHFR. Shorter Debye lengths favor the interaction of the FA Au NPs with
Au NPs, electrostatic interaction of the FA Au NPs with hDHFR may the surface-bound hDHFR.
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buffers, for which the SPR response is very low despite high hDHFR
activity, a short Debye length, and a high aggregation constant. AFM
images acquired for SPR sensors exposed to folate anion-functionalized
AuNPs (with 0 or 100 nM MTX) revealed no significant aggregation of
the AuNPs (see ESI,† Fig. S3). A high surface concentration of isolated
AuNPs was clearly observed on the SPR sensor at 0 nM MTX. For
sensors exposed to 100 nM MTX, the surface concentration of AuNPs
decreased significantly. While the presence of a few closely located
AuNPs was observed at 100 nM MTX, it is clearly insufficient to
conclude that aggregation contributes significantly to the larger SPR
response observed under short Debye length conditions.
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interaction with surface-bound hDHFR. The Henderson–Hasselbach
equation reveals that about 95% of the acidic groups of folic acid are
deprotonated at pH 8, which drops to 15% at pH 6. Thus, the negative
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1
1
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1
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2
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3
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950 | Chem. Commun., 2014, 50, 4947--4950
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