CA2271179A1 - Process for monitoring and detecting small molecule - biomolecule interactions - Google Patents

Abstract

Small molecules (molecular weight less than about 2800) are screened for interaction with biomolecules by contacting a solution containing the small molecule with immobilized biomolecules under test, and generating acoustic waves in the solution using an acoustic wave device. Interaction of the small molecules with the immobilized biomolecules causes a change in frequency of the acoustic waves, which can be detected and analyzed electronically to monitor the interaction.

Description

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

-2-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

-3-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

-4-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 -$-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.

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.

_7_ 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.

_$_ 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 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 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 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 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 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.

Claims