Supporting Information
Title: Nanopore back titration analysis of dipicolinic acid
Author: Yujing Han, Shuo Zhou, Liang Wang, and Xiyun Guan*
Author affiliation:
Department of Biological and Chemical Sciences, Illinois Institute
of Technology, 3101 S Dearborn St, Chicago, IL 60616, USA
*Correspondence:
Dr. Xiyun Guan
Department of Biological and Chemical Sciences
Illinois Institute of Technology
3101 S Dearborn St, Chicago, IL 60616, USA
Tel: 312-567-8922; Fax: 312-567-3494
E-mail: xguan5@iit.edu
Experimental Section
Materials and Reagents. Except benzoic acid (99.5%), which was obtained from Fisher
Scientific (Fair Lawn, NJ), all the other chemicals, including DTPMPA, Tb(NO3)3•6H2O
(99.999%), 2,6-pyridinedicarboxylic acid (99.999%), EDTA (BioUltra grade, 99%),
phthalic acid (ACS reagent, 99.5%), 2,3-pyridinedicarboxylic acid (BioUltra grade,
99%), NaCl (99.999%), HCl (ACS reagent, <1 ppm heavy metals), and Trizma base
(BioUltra grade, >99.8%) were obtained from Sigma-Aldrich (St. Louis, MO). All the
chemicals were dissolved in HPLC-grade water (ChromAR, Mallinckrodt Baker). The
stock solutions of DTPMPA, Tb(NO3)3•6H2O, 2,6-pyridinedicarboxylic acid, EDTA,
phthalic acid, 2,3-pyridinedicarboxylic acid, and benzoic acid were prepared at
concentrations of 1.0 mM each. The electrolyte solution used in this work contained 1.0
M NaCl and 10 mM Tris, with the pH of the solutions adjusted to 7.5 using HCl. Lipid
1,2- diphytanoylphosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster,
AL). Teflon film was purchased from Goodfellow (Malvern, PA).
Protein production. Preparation and formation of mutant α-hemolysin protein pores
have been described elsewhere [S1]. Briefly, the mutant α-hemolysin M113K gene was
constructed by site-directed mutagenesis (Mutagenex, Piscataway, NJ). The mutant αhemolysin monomers were first synthesized via in vitro transcription and translation
(IVTT) using the E. Coli T7 S30 extract system for circular DNA from Promega
(Madison, WI). Subsequently, they were assembled into homo-heptamers by addition of
rabbit red cell membranes and incubation for 1 hour. The heptamers were purified by
SDS-polyacrylamide gel electrophoresis and stored in aliquots at -80°C [S2].
Planar lipid bilayer experiments. Single channel recordings were performed in a teflon
chamber that was divided into two compartments by a teflon film. A lipid bilayer of 1,2diphytanoyl-sn-Glycero-3-phosphacholine was formed on the aperture (150 μm) in the
teflon film (25 μm thick) using the Montal-Mueller method [S3]. The experiments were
carried out at 25 ± 1 °C under symmetrical buffer conditions with 2.0 ml solution
containing 1 M NaCl and 10 mM Tris (pH 7.5) unless otherwise stated. Both the α-
S2
hemolysin protein and the analytes were added to the cis compartment that was
connected to the “ground”. The applied potential was +40 mV. The final concentration
of the α-hemolysin proteins used for the single channel insertion was 0.2–2.0 ng·mL-1.
The generated currents were recorded with a patch clamp amplifier (Axopatch 200B,
Molecular Devices, Sunnyvale, CA, USA). They were low-pass filtered with a built-in
four-pole Bessel filter at 5 kHz and sampled at 20 kHz by a computer equipped with a
Digidata 1440 A/D converter (Molecular Devices).
Data Analysis. All the data were analyzed with pClamp 10.3 (Molecular Devices),
SigmaPlot 12.0 (Systat Software Inc., San Jose, CA), and Origin 8.0 (Microcal,
Northampton, MA). Conductance values were obtained from the amplitude histograms
after the peaks were fit to Gaussian functions. The values of τ off was obtained from the
residence time histograms by fitting the distributions to single exponential functions by
the Levenberg-Marquardt procedure [S4].
Each single-channel current trace was
recorded for at least 1 minute. All the results were reported as mean values ± standard
deviations.
S3
Calculation of Kf values for Tb3+ complexes
A two-step procedure was used to determine the formation constants (Kf ) for various
Tb3+ complexes.
The first step involved the calculation of the Kf value for Tb3+-DTPMPA. Although the
interaction between Tb3+ and DTPMPA or other ligands might produce multi-chelation
complexes, for convenience, the Kf values obtained in this work were based on the
formation of 1:1 (metal : ligand) chelates. To obtain the formation constant for the Tb3+DTPMPA complex, the DTPMPA event frequencies (f1 and f2, respectively) for a single
DTPMPA (2 µM) standard solution (Fig. 1a), and a mixture solution of 2 µM DTPMPA
and 4 µM Tb3+ (Fig. 1c) were first calculated. Then, the ratio (f2/f1) of the DTPMPA
event frequencies was used to determine the concentration of DTPMPA in the mixture
solution. After calculating the equilibrium concentrations of Tb3+ and Tb3+-DTPMPA in
the mixture, the formation constant (Kf-1) for Tb3+-DTPMPA was obtained.
The second step involved the calculation of the equilibrium constant (Kf-2) of the
competition reaction between various ligands and the Tb3+-DTPMPA complex. Take
DPA for example, the following reaction was used to determine Kf-2:
Tb3+-DTPMPA + DPA
Tb3+-DPA + DTPMPA
For this purpose, the DTPMPA event frequency (f3) for a mixture solution of 2 µM
DTPMPA, 4 µM Tb3+, and 2 µM DPA (Fig. 1d) was calculated. Then, the ratio (f3/f1) of
the DTPMPA event frequencies was used to calculate the concentration of DTPMPA in
the solution mixture. After obtaining the concentrations for other species, Kf-2 could be
determined.
With the values of Kf-1 and Kf-2, the equilibrium constant (Kf-3) for the formation of the
Tb3+-DPA complex was able to be calculated using equation Kf-3 = Kf-1× Kf-2. Similarly,
the formation constants for other Tb3+ complexes could be determined.
Supporting References
(S1)
Song, L. Z., Hobaugh, M. R., Shustak, C., Cheley, S., Bayley, H., Gouaux, J.
E., Science 1996, 274, 1859-1866.
(S2)
Zhao, Q. , Jayawardhana, D. A., Wang, D., Guan, X., J. Phys. Chem. B 2009,
113, 3572-3578.
(S3)
Montal, M., Mueller, P., Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3561-3566.
(S4)
Wang, G., Zhao, Q., Kang, X., Guan, X., J. Phys. Chem. B. 2013, 117, 47634769.
S4
Figure S1. Structures of (Left) DPA and (Right) DTPMPA
S5
(a)
(b)
1600
3000
τoff = 6.1
τoff = 0.28
0.2 ms
Count (N)
Count (N)
1200
800
0.01 ms
2000
1000
400
0
0
0
10
20
Residence Time (ms)
30
0
1
2
3
4
Residence Time (ms)
Figure S2. Residence time plots of (a) long-lived events, and (b) short-lived events. The
experiments were performed with the α-hemolysin (M113K)7 pore at +40 mV in the
presence of 10 μM DTPMPA and 5 μM Tb3+. The mean residence time of the long-lived
events was similar to that of DTPMPA alone, suggesting that these events were attributed
to the free DTPMPA in the solution mixture. It is apparent that the short-lived events
were due to Tb3+-DTPMPA complexes.
S6
-1
Event Frequency (s )
102
101
100
10-1
0
0 101
102
103
104
Concentration of DTPMPA (nM)
Figure S3. Dose-response curve for DTPMPA detection. The experiments were
performed in the mutant α-hemolysin protein (M113K)7 pore at +30 mV (cis at ground)
in a solution containing 1 M NaCl, and 10 mM Tris·HCl (pH 7.5). Both the (M113K)7
protein and DTPMPA were added in the cis side of the chamber device.
S7