Download Report

Journal of the Association for Laboratory
Automation
http://jla.sagepub.com/
Backscattering Interferometry for Low Sample Consumption Molecular Interaction Screening
Amanda Kussrow, Carolyn S. Enders, Ereny F. Morcos and Darryl J. Bornhop
Journal of Laboratory Automation 2009 14: 341
DOI: 10.1016/j.jala.2009.08.006
The online version of this article can be found at:
http://jla.sagepub.com/content/14/6/341
Published by:
http://www.sagepublications.com
On behalf of:
Society for Laboratory Automation and Screening
Additional services and information for Journal of the Association for Laboratory Automation can be found at:
Email Alerts: http://jla.sagepub.com/cgi/alerts
Subscriptions: http://jla.sagepub.com/subscriptions
Reprints: http://www.sagepub.com/journalsReprints.nav
Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Dec 1, 2009
What is This?
Downloaded from jla.sagepub.com by guest on October 6, 2014
Original Report
Backscattering Interferometry
for Low Sample Consumption
Molecular Interaction
Screening
Darryl J. Bornhop, Ph.D.
Vanderbilt University
Nashville, TN
Amanda Kussrow, Carolyn S. Enders, Ereny F. Morcos, and Darryl J. Bornhop*
Department of Chemistry and Vanderbilt Institute of Chemical Biology,
Vanderbilt University,
Nashville, TN
Keywords:
backscattering
interferometry,
molecular
interactions,
free solution,
binding affinity,
label free
ackscattering interferometry (BSI), which uses
a simple optical train comprising a HeeNe laser,
a microfluidic channel, and a position sensor, has now
enabled the measurement of both tethered and freesolution, label-free, molecular interactions within just
nanoliters of sample. The simple macro-to-micro interface
allows for a highly efficient assay work flow, which has
been used to interrogate molecular binding interactions
between proteins, ions and protein, and small molecules
and proteins, with a high dynamic range of dissociation
constants (KD) and unmatched sensitivity. With this
technique, the equilibrium KD for several different binding
partners was determined, typically using just
picomoleemicromole quantities of the binding pair at
physiologically relevant concentrations. ( JALA
2009;14:341–7)
B
INTERFEROMETRY
FOR QUANTIFYING
MOLECULAR INTERACTIONS
When two or more light waves are superimposed, an
interference pattern is created. By studying these
*Correspondence: Darryl J. Bornhop, Ph.D., Department of
Chemistry and Vanderbilt Institute of Chemical Biology,
Vanderbilt University, 7330 Stevenson Center, Nashville, TN
37235; Phone: þ1.615.322.4404; Fax: þ1.615.322.2861; E-mail:
darryl.bornhop@vanderbilt.edu
1535-5535/$36.00
Copyright
c
2009 by The Association for Laboratory Automation
doi:10.1016/j.jala.2009.08.006
patterns, the properties of the light waves and of
the material that they have been in contact with
can be explored. The ﬁeld of research known as
interferometry has led to the development of some
of the most sensitive optical techniques available
and has found applications in astronomy, microscopy, oceanography, metrology, seismology, and
molecular interaction studies.1,2 Conducting interaction studies with precision and efﬁciency is fundamental to the optimization of screening assays
because these interactions are the groundwork for
virtually all biological function. Although several
different types of interferometers have been successfully used to study molecular interactions (e.g.,
Mach Zehnder), to our knowledge backscattering
interferometry (BSI) is currently the only technology
that facilitates both heterogeneous (tethered)3,4 and
homogeneous (free-solution) assays,3,5 without
sacriﬁcing sensitivity. BSI has detection limits that
are one to two decades better than other label-free
techniques [e.g., isothermal calorimetry (ITC), surface plasmon resonance (SPR)]. In basic principle,
interferometry measures the change in refractive index (RI), which is the speed of light in the medium,
between the sample and the reference optical path.
The RI is a complicated function dependent on the
molecular dipole of the species in solution and the
composition of the solvent. Structure, hydration
level, and other second-order parameters dictate
the magnitude of the molecular dipole of
Downloaded from jla.sagepub.com by guest on October 6, 2014
JALA December 2009
341
Original Report
a compound. When two molecules bind or a ligand interacts
with a complex, the result is a new compound or complex
with an entirely different RI (molecular dipole) arising from
changes in the level of hydration, structure, and other properties. In short, unexpectedly and nonintuitively, the RI of
the new compound is measurably different than the sum or
difference of the RI for the starting interacting components.
BSI is unique among RI-based interaction sensors (others include SPR, waveguiding, and diffraction) because it interrogates the RI of the entire solution within the channel
(including surface bound species) instead of only quantifying
the ﬁlm thickness at a surface. In addition to this advantage
of bulk-solution interrogation, BSI also has a multipass sensitivity enhancement factor resulting from the characteristic
interaction between the laser and the microﬂuidic channel,
which operates as an optical resonance cavity. In combination, these features have allowed the detection of molecular
interactions and conformational changes in free solution.5
Furthermore, this unique tool has allowed the quantiﬁcation
of DNA hybridization4 at pM sensitivity and interactions
involving extremely small mass changes, such as a sugar
binding to a tethered 100-kDa lectin in the heterogeneous
format.6
THE
BSI INSTRUMENTATION
BSI is a unique interferometric optical approach, in that the
only necessary optic is the channel in the microﬂuidic chip.
This characteristic allows for the use of a very simple optical
train, depicted in Figure 1. In short, the instrument consists
of a red heliumeneon laser (l ¼ 632.8 nm) that illuminates
the microﬂuidic channel, a mirror to redirect the backscattered light, and a camera for transduction of the signal
contained in the fringe pattern. This straightforward, unique
approach to interferometry results in the relatively simple
instrumental conﬁguration used by BSI and is remarkable
in light of the high sensitivity it achieves. Using a proprietary
sample introduction interface, a relatively small volume of
the sample (a few 100 nL to 1 mL) is easily pipetted into
the channel with minimal sample loss. On changing the RI
of the sample via calibration solutions or by introducing
the reacting species, the interference fringes move as depicted
in Figure 2A. As shown in the intensity versus position trace
(Fig. 2B), the BSI signal is found in the spatial output of the
Figure 1. Block diagram of backscattering interferometry illustrating the simple optical train.
camera. Using a proprietary position-sensing algorithm, the
position of the fringes is monitored in real time at ultra-high
resolution (10s of nanometers) and displayed as a BSI signal
versus time trace. This output can be used to quantify the
magnitude of BSI signal, which is related to the RI response
to a change in solute concentration or to a binding event.
Sample analysis time is approximately 30 s per sample.
Future automation of BSI instrumentation with multichannel, automated plate reading capabilities is expected to
provide high-throughput screening of over eight samples
per minute.
FREE-SOLUTION
BINDING ASSAYS
BSI can be used to characterize binding interactions using
either real-time kinetics with on-chip mixing5 or by off-line
mixing with equilibrium end-point determinations of KD.
We have recently shown that a wealth of information may
be obtained using either method by studying a variety of
different binding systems. Here, we present a few representative examples illustrating the broad applicability of BSI for
molecular interaction studies.
KINETIC
ASSAYS
Building on our observations with calmodulin, immunoglobulin G (IgG) e Protein A and interleukin-2 (IL-2),5 we set
out to further benchmark BSI and attempt to show its application as a screening tool for evaluating the inﬂuence of point
Figure 2. (A) Photographs of the shift of the fringes as a result of a refractive index (RI) change. (B) The intensity versus position trace of the
fringes illustrating that the signal is found in the spatial output of the camera. (C) The resulting signal due to a RI-induced fringe shift.
342
JALA
December 2009
Downloaded from jla.sagepub.com by guest on October 6, 2014
Original Report
Figure 3. Kinetics of aB-D3 (10 mM) binding to multiple concentrations of (A) T4L-D70N (1, 2.5, 5, 10, 20, 30, 40, 60, and 90 mM) and
(B) T4L-L99A-A130S (1, 2.5, 5, 10, 15, 20, 40, 60, 80, and 100 mM) were monitored by backscattering interferometry (BSI) (black) with
kinetic traces fit via global analysis (red). Analysis of the steady-state data (C) shows that the magnitude of binding as detected by BSI for RBD3 T4L-L99A-A130S is significantly greater than that seen with RB-D3 T4L-D70N. As a control, RB-D3 was assayed against multiple
concentrations of WT-T4L, exhibiting no binding across the concentration range. (D) The structure of T4L highlighting the sites of
destabilization.
mutations on binding. To do so, we chose a chaperoneeprotein binding system important in the process of cataract formation.7 The small heat-shock protein a-crystallin makes up
nearly half of the protein mass of the eye and also acts as
a molecular chaperone.7 Molecular chaperones help to guide
misfolded proteins back to their native state or to a pathway
that will break them down.8 This process helps to prevent aggregation of the misfolded proteins and, in the case of the acrystallin, to help prevent cataracts. As such, it is important
to be able to quantify how ‘‘destabilized’’ a protein becomes
when it has undergone a transformation, such as a mutation.
BSI was used to monitor the binding of a-Crystallin with
various destabilized mutants of the T4 lysozyme (T4L). For
these experiments, the concentration of the a-Crystallin
aB-D3 was held constant, whereas the concentration of the
T4L mutant was varied. The concentration range for T4LD70N was 1e90 mM, and T4L-L99A-A130S concentrations
ranged from 1 to 100 mM. For a control, wild-type T4L
was used with concentrations ranging from 1 to 100 mM. Figure 3 includes the real-time kinetic data characterizing the interactions of this crystallin proteinechaperone binding
system, steady-state results, and T4L protein structure showing mutated sites. The time-dependent traces were then ﬁt
with a global analysis approach to characterize the interactions; using this sophisticated data analysis algorithm allowed us to extract a quantitative and detailed biophysical
understanding for this system. The results illustrate the selectivity of the a-Crystallin to preferentially bind mutants according to their instability, measured by the energy of
unfolding (DGunf) using ITC. For example, the D70N version exhibits a DGunf of 6.8 kcal/mol with KD1 determined
by BSI to be 0.595 mM, whereas the L99A-A130S mutant
has a DGunf of 3.5 kcal/mol with the KD1 determined to be
0.132 mM.9 Steady-state data of the binding reiterated these
results, showing decreased binding for more stable mutants
of the T4L. It is our belief that this work shows that BSI
can potentially be used to verify some commonly held beliefs
about important pathological mechanisms, for example, we
can quickly and quantitatively measure the inﬂuence of a single (hidden or even possibly buried as with the D70N and
L99A) mutation on molecular interactions at physiologically
relevant concentrations, label-free and in the solution phase.
END-POINT
ASSAYS
Here, we demonstrate that BSI end-point experiments,
conducted using solutions that have been premixed and
equilibrated oﬀ line, can facilitate accurate KD determinations. In these experiments, the ligand or control (nonbinding
ligand) is combined at successively increasing concentrations
with a receptor of ﬁxed concentration. Once combined, the
samples are allowed to equilibrate, followed by introduction
Downloaded from jla.sagepub.com by guest on October 6, 2014
JALA
December 2009 343
Original Report
Figure 4. Binding of carbonic anhydrase (10 nM) to varying concentrations of benzene sulfonamide (0, 0.078, 0.156, 0.312,
0.625, 1.25, 2.5, and 5 mM), performed in DMSO buffer. Error
bars represent the three trial standard deviations.
into the BSI instrument. Although it may not provide the
rich biophysical information afforded by the real-time kinetics experiments, the end-point determination has value, in
that it facilitates very rapid screening and eliminates nonspeciﬁc binding contributions to KD from tethered approaches;
this approach can be used to determine simply if a pair interact, and, if properly used, will provide a quantitative measure
of actual versus calculated equilibrium KD. Interestingly,
with the caveat that the matrix for the interacting species
be constrained or compensated for, no prior knowledge is
needed to access afﬁnity.
Example I
To evaluate BSI’s utility for making end-point binding
aﬃnity determinations, we ﬁrst studied the interaction of carbonic anhydrase (CAII) with an inhibitor. As this determination was performed in DMSO buﬀer, this assay also allowed
the assessment of BSI performance with solvent systems of
great interest to the drug discovery and screening community
Figure 6. The binding affinity of a complementary DNA strand
(5, 50, 500 nM, and 5 mM) to an immobilized target over a multiple
complementary strand. The complementary strand is also
compared with a 3-bp mismatch and control strands at high
concentration (5 mM). All probe strands were allowed for
20 min for hybridization. The control signal is shown at 10 times
its actual signal to allow visualization.
and under conditions considerably more challenging than
with simple buﬀer solutions.
CAII is an enzyme responsible for the conversion of
carbon dioxide to bicarbonate, and inhibitors of this enzyme
are used to treat glaucoma and epilepsy; some drugs targeting CAII may lead to treatments of cancer and obesity.10
BSI was used to perform end-point binding assays on CAII
and ﬁve small inhibitor molecules in solutions containing at
least 1% DMSO (unpublished results). The addition of nonaqueous solvents to a buffer system often complicates an experiment and makes accurate measurements more difﬁcult to
obtain. The ability of BSI to perform such measurements
without any modiﬁcations illustrates its adaptability. These
studies mirrored those performed by SPR11,12 of this wellcharacterized enzyme-inhibitor system. Figure 4 depicts the
binding curve for CAII with benzene sulfonamide and is
quite representative for all the inhibitor species studied.
Error bars in the ﬁgure show the reproducibility for
performing the entire assay in triplicate, whereas the inset
shows the low concentration region. The control, benzene
sulfonamide without CAII, produced a linear response over
the concentration range (not shown) and was used to
normalize the values plotted and used in the determination
of KD. From this assay, we determined KD to be
0.57 0.07 mM compared with the value reported in the literature of 0.80 0.28 mM.11,12
Example II
Figure 5. Binding between p24 (1 nM) and varying concentrations of p24-MAb (0, 50, 100, 200, 400, and 800 nM). Error bars
represent the noise in the measurement.
344
JALA
December 2009
Recognizing that a need exists for improved diagnostic
methods, we have forged a collaboration with the sexually
transmitted disease division of the Centers for Disease Control and Prevention (CDC). Through this association, we
Downloaded from jla.sagepub.com by guest on October 6, 2014
Original Report
Figure 7. (A) The chemical process used to immobilize concanavalin A (conA) onto the surface of the channel. (B) The binding mannose
(0, 10, 20, 40, 60, 80, and 100 mM) to conA with galactose (0, 10, 20, 40, 60, 80, and 100 mM) acts as the nonbinding control. Error bars
represent the three trial standard deviation. (C) The unbound (green) and bound (blue) ribbon structures of conA.
have begun to study the potential for BSI to be used for disease diagnostics. To do so, work has begun on the detection
of antibodyeantigen interactions, with the ultimate goals
being the quantiﬁcation of infection via antibody titer
strength or viral load and the ability to perform therapeutic
eﬃcacy monitoring.
P24 is a core protein of human immunodeﬁciency virus
(HIV) that can be found in the blood serum during the acute
phase (earliest stage) of HIV infection, and therefore may serve
as one of the earliest serological indicators of the disease.13
Serum levels of this antigen decrease as the disease progresses.14 Consequently, the quantiﬁcation of p24 is currently
being explored as an early detection and/or prognostic tool
for HIV infection. Here, we constructed end-point saturation
binding curves using BSI measurements to evaluate the afﬁnity
of p24 for the CDC-developed antiep24-MAb. In this assay,
p24 was held constant at 1 nM, and anti-p24 concentrations
were varied from 0 to 800 nM. From the saturation isotherm
plot (Fig. 5), we calculate for the ﬁrst time the KD for this antigeneantibody pair to have a value of 77.1 16.7 nM
(R2 ¼ 0.99). Notably, no afﬁnity information was previously
known for this binding system because of the fact that the
antibody was newly synthesized and was thus far uncharacterized, highlighting the distinct advantage that BSI can be used
to make novel KD determinations. Results suggest that the
new MAb does indeed bind with the p24 antigen with nanomolar afﬁnity; comparing these results with BSI data collected for
other anti-p24 antibodies may serve as a screening approach
for predicting the relative performance of these synthetic
antibodies in diagnostic assays for HIV.
TETHERED
OR HETEROGENEOUS BINDING ASSAYS WITH
BSI
A unique characteristic of BSI is that the platform can be
used for either homogeneous (free-solution) or heterogeneous (tethered) assays. The Bornhop research group demonstrated the utility of BSI for tethered assays in two
reports,3,4 showing reversible Protein-AeIgG interaction
studies and illustrating the detection of DNA hybridization.
The DNA hybridization experiments were performed by ﬁrst
immobilizing a biotinylated 30-mer of mouse actin DNA
onto the surface of the channel. The unlabeled complementary strand was then introduced into the channel, and the
Downloaded from jla.sagepub.com by guest on October 6, 2014
JALA
December 2009 345
Original Report
BSI signal was measured after 20 min to allow for hybridization. The complementary strand was removed with a sodium
hydroxide wash, and the experiment was repeated. A DNA
strand with a three-base pair mismatch (at positions 5, 15,
and 25) showed signiﬁcantly reduced binding, and noncomplementary DNA strand was used as a control (Fig. 6). This
result demonstrates the utility of BSI to act as a screening
tool for an array of different sequences without needing to
perturb the binding with signaling moieties.
We have since expanded on these preliminary observations substantially, using an extravidin-biotinylated surface
chemistry (Fig. 7A) to study small molecule (monosaccharide)econcanavalin A (conA) interactions and to quantify
multimeric binding or polyvalency with sugar-coated dendrimer or viral particles.6 A representative binding curve
(Fig. 7B) shows that the capture of mannose by conA can
be quantiﬁed, and a saturation-binding curve provides
a quantitative measure of afﬁnity (KD ¼ 44.5 6.2 mM). In
this assay, galactose serves as the nonbinding control. This
study is signiﬁcant for many reasons, particularly because
it demonstrates that BSI uniquely allows quantiﬁcation of
a binding event for just one part in 1000 change in mass,
allowing the protein to be tethered and the small molecule
to be titrated. To our knowledge, this small moleculeeprotein assay has never been performed in this manner on
another platform and represents a challenging assay for
widely used label-free techniques, such as SPR.
Preliminary modeling of the BSI signal has shown a correlation between the strength of the BSI signal and changes
in the structure of the molecule of interest. Interestingly,
unlike other systems we have studied,5 the conformation
of conA (Fig. 6C) does not change appreciably in its overall structure upon binding of its sugar ligand. This phenomenon is depicted in Figure 7C, where the sugar is
shown bound within a pocket, but the lectin structure
has not dramatically changed (green is the unbound structure and blue is the bound form of conA). Despite the minimal change in structure, we can readily detect the binding
interaction and quantify the afﬁnity. Therefore, another
factor (i.e., water of hydration) other than conformational
change must also contribute to the binding signal. These
effects are currently under investigation by more extensive
modeling experiments.
urine (data not shown). Here, we have demonstrated that
BSI can be used in these various formats to characterize
a number of appreciably different binding interactions and
believe BSI to be a promising tool for studying molecular interactions across a signiﬁcantly wide range of drug discovery,
biochemical application, and biomedical application.
ACKNOWLEDGMENTS
This research was supported in part by a grant from the National Institutes
for Health (R01 EB003537-01A2) and the Vanderbilt University institute of
Chemical Biology. Thanks to Tim Granade, and Drs. Ronald Ballard and
David Cox of the CDC for supplying the HIV antibody. M.G. Finn and
Eiton Kaltgrad of Scripps Institute supplied the lectins used in the tethered
interactions assay.
REFERENCES
1. Fan, X. D.; White, I. M.; Shopoua, S. I.; Zhu, H. Y.; Suter, J. D.; Sun,
Y. Z. Sensitive optical biosensors for unlabeled targets: a review. Anal.
Chim. Acta 2008, 620, 8e26.
2. Shtengel, G.; Galbraith, J. A.; Galbraith, C. G.; Lippincott-Schwartz, J.;
Gillette, J. M.; Manley, S.; Sougrat, R.; Waterman, C. M.; Kanchanawong, P.; Davidson, M. W.; Fetter, R. D.; Hess, H. F. Interferometric
ﬂuorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 3125e3130.
3. Markov, D. A.; Swinney, K.; Bornhop, D. J. Label-free molecular interaction determinations with nanoscale interferometry. J. Am. Chem. Soc.
2004, 126, 16659e16664.
4. Latham, J. C.; Markov, D. A.; Sorensen, H. S.; Bornhop, D. J.
Photobiotin surface chemistry improves label-free interferometric
sensing of biochemical interactions. Angew. Chem. Int. Ed. 2006,
45, 955e958.
5. Bornhop, D. J.; Latham, J. C.; Kussrow, A.; Markov, D. A.; Jones, R.
D.; Sorensen, H. S. Free-solution, label-free molecular interactions studied by back-scattering interferometry. Science 2007, 317, 1732e1736.
6. Kussrow, A.; Kaltgrad, E.; Wolfenden, M. L.; Cloninger, M. J.; Finn,
M. G.; Bornhop, D. J. Measurement of monovalent and polyvalent carbohydrate-lectin binding by back-scattering interferometry. Anal. Chem.
2009, 81, 4889e4897.
7. Horwitz, J. The function of alpha-crystallin in vision. Semin. Cell Dev.
Biol. 2000, 11, 53e60.
8. Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo,
J. S.; Taddei, N.; Ramponi, G.; Dobson, C. M.; Stefani, M. Inherent
toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002, 416, 507e511.
9. Latham, J. C.; Stein, R. A.; Bornhop, D. J.; Mchaourab, H. S.
SUMMARY
Free-solution label-free detection of alpha-crystallin chaperone inter-
BSI capitalizes on a simple, unique optical train and the
intrinsic universal high sensitivity of interferometry to serve
as a molecular interaction platform for quantifying binding
aﬃnities over a large dynamic range in KD, in free-solution
or tethered mode, and requiring just picograms to nanograms of the sample. Of great interest to screening applications is the advantage that little or no a priori information
about the system is needed to screen for interaction. Furthermore, it is possible to use BSI with complex matrices, such as
cell-free media,5 DMSO buffer (shown here), serum, and
346
JALA
December 2009
actions by back-scattering interferometry. Anal. Chem. 2009, 81,
1865e1871.
10. Cecchi, A.; Taylor, S. D.; Liu, Y.; Hill, B.; Vullo, D.; Scozzafava, A.;
Supuran, C. T. Carbonic anhydrase inhibitors: inhibition of the human
isozymes I, II, VA, and IX with a library of substituted diﬂuoromethanesulfonamides. Bioorg. Med. Chem. Lett. 2005, 15, 5192e5196.
11. Papalia, G. A.; Leavitt, S.; Bynum, M. A.; Katsamba, P. S.; Wilton,
R.; Qiu, H. W.; Steukers, M.; Wang, S. M.; Bindu, L.; Phogat, S.;
Giannetti, A. M.; Ryan, T. E.; Pudlak, V. A.; Matusiewicz, K.; Michelson, K. M.; Nowakowski, A.; Pham-Baginski, A.; Brooks, J.;
Downloaded from jla.sagepub.com by guest on October 6, 2014
Original Report
Tieman, B. C.; Bruce, B. D.; Vaughn, M.; Baksh, M.; Cho, Y. H.; De
Wit, M.; Smets, A.; Vandersmissen, J.; Michiels, L.; Myszka, D. G.
Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology. Anal. Biochem. 2006, 359, 94e105.
determined by surface- and solution-based biophysical methods. Protein
Sci. 2002, 11, 1017e1025.
13. Iweala, O. I. HIV diagnostic tests: an overview. Contraception 2004, 70,
141e147.
14. Bhardwaj, D.; Bhatt, S.; Khamar, B. M.; Modi, R. I.; Ghosh, P. K.
12. Day, Y. S. N.; Baird, C. L.; Rich, R. L.; Myszka, D. G. Direct comparison of binding equilibrium, thermodynamic, and rate constants
Recombinant HIV-1 p24 protein: cloning, expression, puriﬁcation and
use in the development of ELISA kits. Curr. Sci. 2006, 91, 913e917.
Downloaded from jla.sagepub.com by guest on October 6, 2014
JALA
December 2009 347