Note: Descriptions are shown in the official language in which they were submitted.
CA 02271179 1999-OS-OS
PROCESS FOR MONITORING AND DETECTING SMALL
MOLECULE - BIOMOLECULE INTERACTIONS
$ This invention relates to chemical analysis, and
more particularly to methods of analysis, qualitative and
quantitative, of pharmaceutically active components of a
liquid composition ( an analyte), using biosensors
The principle of a biosensor is to transform
biologically interesting phenomena, such as the binding of
a drug molecule with a target biomolecule, into electronic
information which can be more readily accessed and
processed. A typical biosensor comprises a biochemical
component attached to a form of electronic transducer. The
biochemical component, usually comprising proteins or
nucleic acids, is exposed to a particular chemical
compound, and any resulting chemical interaction is
electronically detected by the transducer.
Transducer technology for use in biosensors can
be based on piezoelectric effects, which are changes in
shape or conformation of certain solid crystals when an
electric voltage is applied to them, or, conversely, the
production of an electric voltage when such a solid crystal
is mechanically deformed. If the crystal is used as one of
the components of an "oscillating circuit", the crystal
will determine the oscillation frequency of the whole
circuit. The crystal itself vibrates at a "resonant
frequency "' which is determined by the physical shape and
size of the crystal, among other factors. Quartz is the
most commonly used piezoelectric crystal, although many
others exist. Under suitable conditions, the circuit will
oscillate very accurately at the same frequency, which is
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measured in Hertz (Hz).
Piezoelectric crystals can be used as the basis
of biosensor transducer platform technologies. If any
material is allowed to contact a clean piezoelectric
crystal surface , as the device oscillates while surrounded
by a gas or vacuum, it will change the resonant frequency
of the device. The size of the observed frequency change
can be used to measure the quantity of material which
adhered to the crystal surface.
These devices have been used to analyze liquid
samples for the presence and content of macromolecular
biochemical substances such as nucleic acids by
hybridization thereof to a complementary nucleic acid
immobilized on a quartz crystal forming part of a
piezoelectric circuit. In such an arrangement, a biosensor
transducer platform comprising a platform-like quartz
crystal, a first electrode on its lower surface and a
second electrode on its upper surface, with the immobilized
biomolecule on the second, upper electrode, is used. The
liquid containing the test substance is caused to flow over
the immobilized biomolecule, while the lower electrode and
crystal surface contact gas or vacuum. The resulting
bonding or hybridization of the nucleic acid in the test
solution (analyte) to the immobilized nucleic acid on the
electrode causes a change in the vibrational frequency of
the circuit, as compared with that of the circuit involving
the immobilized nucleic acid itself. The existence and
magnitude of the change of frequency is a measure of the
presence and quantity of the nucleic acid under test, and
can be electronically translated into detection of the
presence and measurements of the quantity of the nucleic
acid under test, in the analyte solution.
a
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A mathematical expression known as the Sauerbrey
equation has been developed, to describe the piezoelectric
effects of substances bound to the piezoelectric crystal.
This predicts that an increase in mass of the substance
bound to the piezoelectric crystal will cause a
proportional change in the frequency of oscillation of the
circuit. It also indicates that, to measure mass changes in
a meaningful way, the change must be of the order of at
least one nanogram (one billionth of a gram), so that the
method would only be useful for measurement of
macromolecules.
There is an ongoing need for improved methods for
detecting and monitoring small molecules, i.e. molecules of
molecular weight 2800 Da or less. This arises, for example,
in the screening of drug candidates, which for the most
part are "small molecules", for activity or binding
affinity with certain target molecules.
The present invention provides a process whereby
biosensors based upon piezoelectric effects and
measurements as described above may be used to detect, to
quantify and to monitor the chemical and biochemical
reactivity and properties of small molecules, i.e. those of
less than about 2,800 Da molecular weight. Contrary to the
theory and predictions derived from the Sauerbrey equation,
frequency changes caused by binding of small molecules to
biomolecules such as nucleic acids and proteins immobilized
on piezoelectric crystals in oscillating circuits are much
larger than would be expected, and, in fact, in the
opposite direction from that predicted. The piezoelectric
crystal-based device when operated with the piezoelectric
crystal in contact with or submerged in liquid is
effectively much more sensitive than the Sauerbrey equation
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suggests. It can effectively measure adhered matter in the
picogram (one thousandth of one billionth of a gram, 10-9
gram) range, making it useful in detection of the adherence
of small molecules to immobilized biomolecules such as
proteins and nucleic acids in such piezoelectric
biosensors.
Thus according to the present invention, in one
aspect, there is provided a process for detecting the
interaction of small molecules with biomolecules, which
comprises contacting a liquid solution suspected of
containing a small molecule of interest with said
biomolecules in a biosensor, said biosensor comprising a
piezoelectric material, an electrode electrically connected
to said piezoelectric material, said biomolecules
immobilized on said electrode, and an electrical circuit
involving the electrode and the piezoelectric material and
having characteristic, measurable electrical output
signals, and monitoring change in at least one of said
electrical output signals caused by interaction of said
small molecule of interest with the immobilized
biomolecules.
It has been found, according to the invention,
that piezoelectric crystals submerged in a liquid, or
contacting a liquid on one surface, no longer obey the
Sauerbrey theory. This theory appears to be true only for
piezoelectric crystals operating in gases or in vacuum. In
a liquid medium, a piezoelectric crystal undergoes a
dramatically fundamental change in the manner it operates
and responds to adhered matter. In fact, the vibrating
crystal transmits ultrasonic or acoustic waves into the
liquid medium. While it is not intended that this invention
should be limited to any particular theory of operation, it
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appears that the liquid closest to the crystal surface can
"slip" along the vibrating surface, so that it is the
viscosity and "stiffness" of the. liquid which is being
probed by the ultrasonic or acoustic waves emitted by the
$ vibrating crystal. In a manner reminiscent of sonar, the
transmitted acoustic waves can couple back to the crystal,
and thereby detect the presence of, and hence changes in
the characteristics of, nearby molecules. The molecules in
fact change the frequency of the acoustic waves. Binding
of or interaction of small molecules with biomolecules
immobilized on or near the crystal causes further changes,
as compared with the immobilized biomolecules themselves..
Since the sensors described herein operate on a
1$ principle of transmitting ultrasonic waves into a liquid
medium, they are termed "acoustic wave biosensors", or
acoustic wave devices, AWDs, and this term is sometimes
used herein to denote such devices. Other acoustic wave
devices which can be used in the present invention, besides
piezoelectric devices, include magnetic activated resonator
sensors (MARS).
In a preferred embodiment of the process of the
invention, the liquid solution suspected of containing the
2$ small molecule of interest is flowed continuously across
the immobilized biomolecules, and measurements of chosen
electrical signals are made continuously as the solution
flows. A variety of different solutions can be flowed
across the biomolecules of the device successively, in a
continuous operation, and measurements correlated to the
different solutions. In this way, screening of a number of
small molecules, e.g. drug candidates, for interaction with
biomolecules such as proteins and nucleic acids, can be
conducted rapidly, relably and economically.
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The electrical signal used as the basis of
measurement of changes caused by the interaction can be any
detectable output which changes as a result of the
interaction. For example, it can be the frequency of
oscillation of the piezoelectric crystal, as detected by
the circuitry. Preferably, however, changes in impedance of
the crystal are used as the basis of measurement. For this,
pulsed electrical power is supplied to the crystal, and the
resolution of the impedance measurements can be improved by
using a selection of different frequencies of the power
input.
A specific type of piezoelectric-based AWD for
use in the present invention is a thickness shear mode
device, TSM.
The preferred process of the present invention
accordingly uses an acoustic wave device (AWD), nucleic
acids immobilized on the AWD, a flow cell which contains
the AWD and which permits the flow of liquids across the
surface of the AWD to which the biomolecules are attached,
a means of sending and receiving electronic signals to and
from the AWD to determine changes in acoustic frequency
associated with small molecule interaction with nucleic
acid or protein targets, a means of storing and processing
the electronic signals collected from the AWD, and a method
to interpret the data and correlate it to the determination
of small molecule interaction affinity for biomolecules.
As noted above, the AWD is a type of biosensor
used as a means of detecting the presence of molecules
dissolved in a liquid medium. An AWD produces and
propagates acoustic waves into a liquid medium.
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Thus from a broad aspect, the present invention
provides a process, and suitable apparatus, for detecting
or monitoring the interaction of small molecules,
especially those of molecular weight 2000 Da or less, with
biomolecules such as nucleic acids, which comprises
immersing immobilized biomolecules under test in a solution
containing the small molecule of interest, generating
acoustic waves in the solution by use of an AWD, and
detecting and analyzing frequency changes in the acoustic
waves attributable to interaction of the small molecules of
interest with the immobilized biomolecules under test.
Typically, the AWD is made of a piezoelectric
material, such as quartz, and is shaped into planar form,
often circular. The device should also be shaped in such a
way as to allow the surfaces to vibrate parallel to the
plane of each face. To each face, metal electrodes are
affixed to allow intimate electrical contact, so that the
piezoelectric effect can occur.
The AWD is made useful for biosensor applications
by attaching or immobilizing biomolecules onto the AWD
surface. It is well known that biomolecules interact very
selectively with other molecules to form aggregate
compounds. By immobilizing a particular biomolecule
species onto the AWD, a very selective biosensing device
can be made.
There are many immobilization protocols described
in the literature. In particular, silane adhesion agents
have been used to attach biomolecules to biosensors,
including piezoelectric AVD's. One specific and highly
effective method is disclosed in International Patent
Application PCT/CA/00969.
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By attaching one end of the biomolecule which is
not involved in small molecule interaction to the surface
of the AWD, the remaining portions of the biomolecule are
free to associate with small molecules dissolved in liquid.
In the preferred embodiment, the AWD is housed in
a flowcell which performs several functions. It protects
the AWD from damage. It allows electrical contact to be
made with the AWD, and allows the electronic signals to
pass from the AWD, through the flowcell, and to the outside
of the flowcell, where the contacts terminate. It also
allows liquid or gas to flow over one or both sides of the
AWD. Each face of the AWD is suitably positioned over a
separate chamber of a pre-determined volume. The liquid
flows into one of the chambers through one port, through
the chamber, and out of the chamber through a second port.
The other chamber is not connected to the liquid supply,
and may be kept sealed or purged with gas. The faces of
the AWD are sealed, typically by using "o-rings".
Liquids can be introduced into the flowcell by
means of a suitable pump, such as peristaltic, syringe, or
piston, so that a continuous flow of liquid passes through
the flow cell. Water is the most commonly used liquid for
this purpose; however, many additives may be dissolved in
the water so as to provide an environment suitable for
measuring biomolecular interactions. Cations (such as
lithium, sodium, potassium, magnesium, calcium, ammonium,
alkyl ammonium, quaternary ammonium, guanidinium), anions
(such a chloride, phosphate, carboxylate, sulfate,
sulfonate, carbonate, borate), buffers (to regulate pH),
solubilizing agents (detergents, surfactants, organic
solvents), chelators (such as EDTA), and anti-
bacterial/anti-microbial substances may be present in the
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water.
In most typical pharmaceutical drug screening
applications, a preferred use of the present invention,
multiple samples of small molecules are required to pass
into the flowcell to evaluate their affinities for the
biomolecule immobilized onto the AWD. The small molecules
are normally stored in separate containers, or can also be
stored as mixtures. The sample concentrations can be either
known or unknown. A known volume of sample is injected
into the flowcell for analysis by means , for example, of a
Rheodyne sample injection valve. The preferred method is
to use a commercially-available "autoinjector" device which
possesses such a valve, and is capable of injecting known
volumes of sample into the continuously flowing liquid,
which then travels through the appropriate tubing to the
flowcell. Each sample is injected sequentially. The
autoinjector method allows multiple samples to be processed
in a predetermined order automatically. The autosampler
"XXL 232" supplied by Gilson is most suitable for this
purpose.
To send and receive electronic signals, the
electrical contacts, which terminate on the outside of the
flowcell, are connected to an appropriate electronic
measurement device which is capable of reading the
particular frequency that the AWD is operating, at a given
interval of time. To do this, the electronic measurement
device should be capable of transmitting electrical power
to the AWD, as well as being capable of reading frequency.
One such method is known as the "network analysis method",
in which a Hewlett-Packard 4395A network/spectrum/impedanc
analyzer is used to characterize the AWD primarily by what
is known as "series resonant frequency". Many other
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parameters such as parallel resonant frequency, phase,
impedance, resistances, capacitances, and inductances may
be used, in an analogous manner. The Hewlett-Packard 4395A
is controlled by a computer program, which may be installed
on a separate computer system, that allows the measurements
to be started at a predetermined time and date, carried out
at predetermined intervals of time throughout the course of
an experiment, and stopped at a predetermined time and
date.
The frequencies, and other electronic parameters,
that are detected by the Hewlett-Packard 4395A, are also
stored as a data file in an appropriate format which allows
the data to be graphed as time vs frequency, or time vs
some other electronic parameter. This allows the magnitude
and/or the area of the peaks present in the data graph to
be determined.
The magnitude and/or the area of the peaks over
time correspond to the relative strength of the small
molecule interaction. If one small molecule sample
generates a greater peak height, and/or peak area signal
compared to a signal generated by a different small
molecule, then the first small molecule can be interpreted
as having a greater affinity for the biomolecule than does
the second small molecule. If both small molecule samples
generate the same signal intensity over time, but the first
sample was known to be more dilute than the second sample,
then the first sample can be interpreted as having a
greater affinity for the biomolecule than does the second
small molecule.
Such a procedure for data analysis can be carried
out automatically using commercially available software
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such as those typically used to process chromatographic
data.
A specific preferred embodiment of the present
S invention is diagrammatically illustrated in the
accompanying drawings, in which:
FIGURE 1 is a diagrammatic top view of a
piezoelectric sensor platform for use in the invention;
FIGURE 2 is a diagrammatic side view thereof;
FIGURE 3 is a diagrammatic side view of the top
electrode of the device with biomolecules immobilized
thereon;
FIGURE 4 is a similar view of the biosensor
mounted in a flowcell.
Figs. 1 and 2 show a quartz substrate 10 having a
top electrode 12 on its top surface and a similar bottom
electrode 14 on its bottom surface, both in electrical
contact with the substrate. The arrows on Fig. 2 indicate
the ability of the substrate to oscillate in the plane of
its surfaces on application of electric power of
appropriate frequencies.
Fig. 3 shows biomolecules 16, e.g. nucleic acids
or proteins; immobilized to the upper surface of the top
electrode 12 through the intermediary of a chemical
immobilizing agent 18, which is suitably a cross-linked
silane optionally including linkers and tethers as
disclosed in aforementioned International Patent
Application PCT/CA98/00969, the disclosure of which is
i
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incorporated herein by reference.
Fig. 4 shows the biosensor 20, including the
substrate 10, electrodes 12, 14 and immobilized
biomolecules 16 inside a flowcell 22 and ready for
operation in the process of the invention. The flowcell 22
has an outer housing 24 inside which is mounted a cell 26
having an upper chamber 28 and a lower chamber 30. The
biosensor 20 is mounted in a seal 32 separating the
chambers 28 and 30, with the top electrode 12 and the
immobilized biomolecules 16 protruding into the upper
chamber 28 and the bottom electrode 14 protruding into the
lower chamber 30. Liquid containing the small molecule of
interest fills the upper chamber 28 and flows continuously
therethrough, from liquid inlet 34 to liquid outlet 36,
both protruding outside the housing 24. Inert gas such as
nitrogen fills the lower chamber 30, and flows continuously
therethrough from gas inlet 38 to gas outlet 40, similarly
protruding outside the housing 24. The use of inert gas in
this manner permits free oscillation of the piezoelectric
substrate, and maintains an inert environment, of
controlled humidity (preferably dry) in contact with the
bottom surface and bottom electrodel4, for increased
reliability of results.
In practical operation, liquid inlet 34 is
connected via suitable pumping arrangements to a multiwell
plate containing a plurality of different liquid solutions
for analysis. The solutions are pumped sequentially through
the upper chamber 28 of the flowcell, while the electrodes
of the substrate are connected to suitable circuitry via
electrical connections 42, 44. Readings of output from the
electrodes are made and suitably displayed continuously, in
real time, as the solutions are flowed through, and
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appropriately recorded for analysis.
Small molecules of molecular weight up to about
2,800 Da are monitored for biomolecule interactions
according to the present invention. One specific example of
such a molecule is the Tat-20 peptide, which interacts with
RNA (TAR) which can be immobilized as the biomolecule in
the present invention. This provides a monitor of HIV
infection
Preferably, however, the method of the invention
is used to screen the activity of small molecules of up to
about 2000 Da molecular weight, for their interaction with
various nucleic acids immobilized on the substrate.
Screening of drug candidates for such interactions is
increasingly important in research and development, in the
pursuit of active small molecules capable of, for example,
inhibiting the activity of viral RNA and other nucleic
acids, such as those found in HIV infected patients. A
particular class of small molecules with which the process
of the invention has been used with notable success is the
class of antibiotics known as aminoglycosides, which
includes such well-known antibiotics as streptomycin,
neomycin and gentamycin. These are highly charged
molecules, which interact with nucleic acids. Accordingly
the method of the invention is particularly suitable for
screening these and other compounds of the same general
family for their interaction with specific nucleic acids.
Another specific example of application of the present
invention is in connection with the toxicity of drug
compounds, in regard to unwanted nucleic acid binding,
which may be minimized by use of the process of the present
invention with such compounds.
