GB2507947A

GB2507947A - Method for molecular affinity testing using flow cell with porous membrane

Info

Publication number: GB2507947A
Application number: GB201216496A
Authority: GB
Inventors: Patrick Douglas Shaw Stewart
Original assignee: Shaw Stewart P D
Current assignee: Shaw Stewart P D
Priority date: 2012-09-14
Filing date: 2012-09-14
Publication date: 2014-05-21
Also published as: GB201216496D0

Abstract

A method of investigating the chemical affinity between one or more target substances and a set of one or more test substances comprises the step of placing a solution of the target substance in a non-flowing chamber 11 of a flow cell that is separated from a flow-through chamber 13 by a semi-permeable or porous membrane 15. In a second step, a carrier fluid that carries discrete batches of one or more test substances in solution is moved through the flow-through chamber. In a third step, the retardation and spreading of the test substances that results from their chemical interactions with the target substance is detected. The target substance may be a macromolecule, protein, peptide, nucleic acid, virus, carbohydrate, lipid, or polysaccharide. Preferably, the time taken for one or more test substances to reach a detector is measured, a longer time indicating a stronger interaction between the target and test substances. Test substances may be passed through the flow cell one by one or in combination.

Description

Method and apparatus for molecular affinity testing

TECHNICAL FIELD

Ihis invention relates to methods of detecting chemical affinity between test substances and target substances. Ihis includes the analysis of the chemical affinity of small irtolecules to biological and organic macromoiccules. The method uses a combination of the approaches of dialysis, inicrotluidics and chromatography. It has applications in mcdical, chemical, biochemical and biological rescarch and analysis, and in drug discovery.

BACKGROUND OF THE INVENTION

Therc arc many ways of detecting weak or strong affinity and binding betwcen small molecules and macromoleculcs, or, spcaldng more gencrally, bctwccn test substances and target substances of any molecular weight. Conilnon approaches include nuclear magnetic resonance (NMR), x-ray and neufton crystallographic analysis, surface plasmon resonance and thermal shift assays (also called differential scanning fluorometry).

A recent approach has involved chemically inmiobilizing a target substance onto a chromatographic column, and passing test substances through the colunm. This chromatographic approach is known as weak affinity chromatography. Like surface plasmon resonance, this approach has, until now, always involved chemically immobilizing the target subsiance. Immobilizing the larget substance is labor intensive, wasteful of materials, and likely to distort the target substance at the molecular level.

All of these established approaches have iniportant disadvantages. For example NMR and crystallography are expensive, slow and difficult, while surface plasmon resonance and weak affinity chromatography require chemical immobilization of the target macromolecule. Thermal shift assays do not work with all targets.

A much simpler and more widely applicable approach. which has some of the advantages of weak affinity chromatography but does not involve the chemical immobilization of the target molecule, is described herein.

BRIEF SUMMARY OF THE INVENTION

The present invention aims to satisfy the long-felt need for devices and methods for detecting chemical affinity that are quick and easy to use, highly sensilive, and compatible with most classes of target molecules, It finds affinity between one or more "target substances" which might be niacroniolecules (such as proteins or nucleic acids) and a set of "test substances" (which might he small molecules). The target substance mmiust have a higher mmiolecular weight than the test substances.

One aspect of the invention is a method of physically immobilizing target substances without using covalent chemical bonds to attach them to a solid material.

A second aspect is a method of detecling spreading or delaying of Ihe passage of a batch of a rest substance as it interacts with a sample of a target substance in solution.

A third aspect is a system for inmiohilizing target substances using a porous membrane.

A fourth aspect is inftoducing a greater degree of auomaion in a single system for molecular affinily testing.

There are many important uses of the invention. For example. it can be used for drug discovery by iiuniobilizing targct protcin inoiccuics in a non-flowing conduit, passing small moiccules (including lead compounds) in solution through a flow-through conduit, and recording the concentration of eluted small molecules. The small molecules used could be "fragments" for drug discovery. (Fragments are small molccules that are too small to he pharmaceuticals, but which can hc chemically grown or combined with other fragments to give sufficiently strong binding and great enough specificity to yield actual pharmaceuticals.) Thc invcntion can also bc uscd in othcr divcrsc rcscarch arcas. including rescarch into nmtcrials.

biochemical research, and chemical research.

BRIEF DESCRIPTION OF THE DRAWINGS

Figurc 1 is a close-up cross-sectional view of a flow-cell that is suitable for the method.

Figure 2 is a cross-section of a similar how-cell, showing a clamp mechanism and a viewing window from the side.

Figure 3 is an exploded view of a similar apparatus with a longer pair of conduits.

Figure 4 is a schematic plan view of a similar apparatus.

Figurc 5 shows a cross-section of the apparatus of figure 4 along the line A-B.

Figure 6 is a schematic plan view of an apparatus with a longer conduit that possesses more turns.

Figurc 7 is a schematic plan view of an alternative approach where the non-flowing and flow-through conduits run at right angles to each other.

Figure S shows a cross-section of the apparatus of figure 7 along the line C-D.

Figure 9 is a schematic plan view of an apparatus that follows an alternative approach where the non-flowing and tlow-through conduits are wide indentations that possess raised supports that define the position of the permeable membrane.

Figure 10 shows a cross-section of the apparatus of figure 9 along the line E-F.

Figure 11 is a schematic view of a system that includes a flow-cell, such as those of the previous figures. associated with several peripheral components including a sampler. sampling valve, pumps.

and a valve for assigning the output of a flow-cell either to waste or to a detector.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes a method of measuring the interactions between a set of test substances and one or more target substances. The test substances are typically random "libraries" of small molecules. ligands, drugs or "fragments" for (hug discovery, hut they can also he peptides, (generally small) proteins, nucleotides, nucleosides, nucleic acids, oligonucleotides, DNA, RNA, coenzymes, cofactors, signaling compounds, agonists, antagonists, inverse agonists, hormones. vitamins, lipids, inhibitors, dyes etc. larget substances can he uiacromolecules including proteins, nucleic acids, carbohydrates, polysaccharides. lipids and their complexes. They can also be non-biological synthetic or naturally occurring compounds. Viruses. microorganisms. synthetic macromolecules etc. can also be used as target substances.

The invention will now he described in detail, with reference to the accompanying drawings. Like numerals denote like components.

Figure 1 shows the essential components of a novel dialysing flow-cell for carrying out the method.

The flow-cell is first primed by introducing the target molecule into the non-flowing conduit 11.

Figure 1 shows the dotted outline of an inlet 12 as a hidden detail. Ihe [low-cell possesses an outlet in a similar position at the opposite side of the device. The target molecule is introduced by injecting it through the inlet while allowing fluid to pass out of the outlet. One or more test substances are then introduced into a flow-through conduit 13 via a second inlet 14 and outlet. (The geometry of the inlets and outlets is not significant.) The non-flowing conduit is separated from the flow-through conduit by a porous or semi-permeable membrane 15. the membrane is selected with a molecular cut-ofi that prevents the passage of the target substances hut allows the passage of the test substances. One simple method of constructing the flow-cell is to trap the porous membrane between an upper 16 and a lower block 17, which define the non-flowing and flow-through conduits respectively. The two blocks can advantageously he clamped or pressed together as described below, or fused or glued to cacti oilier.

The membrane can he clamped between the two blocks as shown or mounted independently (not shown), Figurc 2 is a cross-section of the flow-cell of Figure 1, which shows that the two blocks can be pressed together by means of a base 21 and a clamp 22. If at least one of the blocks 16 is transparent. it is possible to inspect the two conduits visually. Advantageously, a ngid transparent sheet or block 23 is placed over the iransparent block, lithe transparent sheet 23 is glass a compliant sheet (e.g. a rubber sheet) 24 should he placed below the clamp 22 to preveni cracking of the glass. Ihe rigid transparent sheet (e.g. glass) 23 prevents the transparent block 16 from bending. If the transparent block is rigid (e.g. if it is made from glass) then the extra transparent sheet is not necessary. It is helpful but not essential to be able to see into the conduits for dehubbling, detecting precipitation of the target substance etc. The upper and lower blocks can he made from a variety of materials. Many inert metals such as stainless steel or chromium-plated brass are suitable. A variety of plastics can be used. PTFE. PEP and polypropylene arc inert, and the latter two are or can be transparent, which makes them suitable for the upper block (assuming that the conduits are viewed from above, which need not be the case).

Composite materials, ceramics and cement-based materials are also suitable. (ilass can he used and is very suitable for one or both blocks.

Figurc 3 is an exploded view of a similar f1ow-cell. This figure does not show the base. clamp or transparent sheet. It shows an upper block 16, a non-flowing conduit 11 with an associated inlet 12 and outlet 31 for priming it, a porous membrane 15, a lower block 17, and a flow-though conduit 13 with an associated inlet 14 and outlet 32. In order in increase the length of Ike non-Flowing and Flow-through conduits, they have two hairpin bends. By making corresponding bends in both conduits i.e. by making the conduits mirror inmges of each other, the conduits mate and are in communication along their lengths.

Figure 4 is a simplified plan view of the same apparatus that shows only the outline of the upper and lower blocks, and the outline of the conduits. Ihe inlets and outlets and oLher details are not shown.

FigureS shows a cross-section through the upper 16 and lower 17 blocks of figure 4 along the line A-B. This figure shows the non-flowing 11 and flow-through 13 conduits, and the porous membrane 15.

Both conduits are flattened (their heights arc less than their widths) to decrease the distance that the test substances need to diffuse, thereby decreasing the time required for adequate equilibration and allowing the solution containing the test substances to flow faster in die flow-through conduit.

Figure 6 is also a simplified plan view and it shows that the length of the conduiis can he further increased by incorporating many bends.

Figure 7 is a simplified plan view that shows that the non-flowing and flow-through conduiis can cross each oiFer e.g. at right angles. l'he upper (e.g. non-flowing) conduit is shown in grey, while the lower (e.g. flow-through) conduit is shown in black. This approach has the advantage that it is not necessary to position the upper and lower blocks precisely, but it may necessitate slower Ilow-rates because it will typically increase the distances that the test substances must difthse to give adequate equilibration across the permeable membrane. This increased diffusion distance results because the flow-through and non-flowing conduits do not rnae along their entire Iengths Figure S shows a cross-section through the upper 16 and lower 17 blocks of figure 7 along the line C-D. The cross-section passes along one seedon of the lower (e.g. flow-through) conduit 13, and across many seedons of the upper (non-flowing) conduit 11.

Figure 9 is a simplified plan view that shows that the upper and lower conduits can be flattened and widened if supports 91 are included that define the vertical position of the permeable membrane.

Figure 10 shows a cross-section of this arrangement along the line E-F.

Figure 11 shows how the novel flow-cells of the previous figures can he incorporated into a functional system for testing chemical affinity. Upstream of the flow-cell 1 11. a pump 112, piswn, syringe or other fluid-moving device is provided that can move a carrier fluid (described below) through the flow-cell. Downstream of the flow-cell there is a detector 113. A sampler 115. a sampling valve 114, and a second pump 116, piston. syringe or other means of moving fluids is provided in order to inuoduce test substances into the flow-cell as follows: the sampling valve 114 switches to a position where the sampler is connec[ed o die second pump, and test substances are picked up by the sampler and nxwed into the valve. Afler this the valve is turned unlil the lest suhsLances are inftoduced into the tluid path that leads from the first pump 112 to the flow-cell 111, so that the test substances can he moved into the flow-cell. through it and into the detector.

Figure 11 shows a sampling valve 114 with four ports, but clearly a six-port valve with a sample loop could he used. this increases the volume of solulion containing test substances lha is introduced into the tluid flow to the flow-cell in each cycle of the valve.

Binding of one or more test substances to the target substances results in those substances passing through the flow-cell more slowly Ihan Ihose that do not hind Effluent from lhe flow-cell passes out of the flow-cell and through a thsliihution valve 117. From this valve it moves either to a detector 113 or to wasw 118. By direchng a proportion of the effluent to waste the flow raw through the flow cell can he greater than the flow rate that the detector (e.g a mass spectrometer) is ahle to accept. A wide variety of detectors can he used that are conventionally used for chromatography. These include mass spectrometers (MS), UV detectors, flame ionization detectors, evaporative light scattering detectors.

refractive index detectors, fluorescence detectors etc. MS is particularly suitable because of its very high sensilivity and because several arge substances can he detected simultaneously. lest substances can he inftoduced singly or in batches (see below). A delector such as MS that can monilor several test substances simultaneously is helpful if the test substances are introduced in batches.

All valves could he rotary valves or slide valves (rotary valves are shown in Figure 11). As an alternadve to pumps eke, gravily can be used o move the fluids. All connecling wbes downstream of the sampling valve 114 o the deedor should he kept as short as possible. Also low-diameter tubes should be used to reduce spreading of peaks due to the tendency for slugs of liquid to become spread out along the tubes. Long tubes and large-diameter tubes will give wider peaks at the detector.

11w carrier fluid winch uiuves test substances through lie flow-cell can in principle be a gas or a liquid. A liquid is most common. If it is a liquid, it could be aqueous or based on organic solvents. If ii is aqueous it can contain sails or buffers to stabilize target substances such as proteins. Suitable salts include volatile salts such as ammonium acetate, which are compatible with MS detectors. If it is a gas, only volatile rest suhsances can he used, and, with appropriafle design. the niemhrane can he omitted. The target substance remains in solution.

Ihe flow-cell works on the following principle: when lest suhsances are moved into the flow-through conduit, a proportion of each suhsance difluses through the permeable membrane into the non-flowing conduit. Since the carrier fluid is moved along the tiow-through conduit and the test substances equilibrate across the membrane in each position, the test substances will spread out in time and space. hut nevertheless they will each move through the flow-cell as a "peak". If the target substance in the non-flowing conduit interacts with a test substance, a proportion of the test substance is effeclively removed from solution in each position. Ibis causes extra test suhslance to diffuse through the permeable membrane, which delays the passage of the interacting test substance in comparison to test substances that do not interact. Moreover further delays may arise because it takes time for the test substances to dissociate from the target substance. The peak of a test substance that interacts is therefore spread out more in time and space. The principle has similarities to both chromalography and dialysis. fly moniloring the concentration of lest substances downstream of lhe flow-cell, test substances that interact with the target substance can he identified.

Note that it is important that the fluid in the flow-cell does not move too fast. It needs to move slowly enough to allow significant equilibration between the test substances across the membrane and between the flow-through and non-flowing conduits in each position. The appropriate flow rate depends on the molecular weights of the test substances, the properties of the membrane and the geometry of the flow-cell. Ihinner conduits allow faster flow rates.

TEMPERATURE CONTROL

An important variation of the invention involves controlling the temperature of the flow-cell and its contents. Ihis can he achieved by insulaling the flow-cell and conlrolling the temperature of lhe solution entering it. Temperature control is helpthl to increase the reproducibility of binding. It can also be used when one or more test substances binds so tightly to the target substance that they are not eluted. The closeness of binding can then be investigated by slowly increasing the temperature while monitoring the solution passing oul of the flow-cell. When a hound suhslanee is released, the temperature in the flow-cell is a measure of the closeness of binding, with higher temperatures generally corresponding to stronger binding. Alternatively, the flow-cell could itself he heated or cooled.

METHOD AND APPARATUS WITH MULTIPLE FLOW-CELLS

Advantageously, two or more flow-cells can he run in parallel. A first flow-cell contains a sample of the target substance in the non-flowing conduit, as described above. A second flow-cell is identical except that it lacks the target substance. Comparisons can then he made between the behaviors of each test substance in the two flow-cells. The differences in their behavior reflect interactions of the target substance with the test substances. Ihis approach is helpful because test suhslances may inleract with other components of the system such as the permeable membrane, the solvent that the target substance is dissolved in, or the walls of the conduits. In one embodiment, each flow-cell has its own detector.

In a second embodiment a switching valve can divert the how from each channel to a single detector in turn. The unused carrier fluid from both flow-cells passes to waste.

SIMULTANEOUS TESTING OF MULTIPLE TEST SUBSTANCES

One approach is to test each test substance or compound in sequence. However a faster approach is to mix batches of several test compounds and test them simultaneously. Since some detectors can analyze the concentrations of multiple compounds simultaneously, this can provide as much information as the sequential approach (or more information, see below). MS is particularly useful for simultaneous measurement.

STRATEGY FOR THE AUTOMATIC ELUCIDATION OF BINDING

PATTERNS

It is helpful to mix a plurality of test compounds together for a different reason. That is, to gain extra information. In drug discovery, for example, it is helpful to find out which fragments or lead compounds bind competitively. For example if two compounds are capable of binding. hut only one binds when both are present simultaneously, this may indicate that these compounds bind to the same site on the targel suhsance. If, on the other hand, hoth compounds hind equally well simulianeously and separately, this nrny imply that they bind to different binding sites. In cases where several test substances can be found that interact with a target substance, it is very helpful to analyze the patterns of their interactions. These patterns can give information about test substances and also about the number and organization of the correspnding binding sites on the target substance, For example two compounds may hind independently. no affecling the hinding of the other, while a third compound blocks the binding of both. One interpretation of this pattern is that the first two compounds bind to sites that are close to each other, and that the third compound blocks both sites. Another explanation is that binding of the third compound causes allosteric rearrangement of the target substance. Further experiments may he required to make this distinction.

For this type of analysis it is helpful to generate a table (or its equivalent in digital information) of many test suhstances showing their interactions, the invention can provide and use such a tahle automatically using software that implements appropnatc algorithms. A simple algorithm is to identify a set of test substances that bind to a target, then to test all pairs selected from this set, recording the effect of combination on the passage of both substances. More complex algorithms can he used for a larger set of hinders where using all the pairs would give too many experimenis. For example. a set ol hinders is identified, and the strongest hinder of these is singled out. Ibis strongest hinder is then tested in combination with all the other binders. This allows the identification of a subset of binders that bind independently from the strongest binder (they arc likely to bind to a different site on the target substance). The strongest binder of this subset is now singled out and is tested in combination with all other binders (except for the original strong binder because there is no point in repeating that experiment). this process is continued until all of the main hinding sites have been identified.

Clearly, many other similar algorithms can provide useful information.

In sonic cases test substances bind so strongly that little or no material is detected at the detector even after long periods. In such cases, competitive binding assays can be performed where a first bound compound is released from the flow-cell by passing through a second conipound that hinds more tightly to the target.

Both simple and competitive or parallel binding experiments are carried out automatically using a sampler that can pick up solutions from a 2-d array of solutions. lest suhstances can he mixed automatically hy the sampler or an automatic fluid handling system, starting with stock solutions ol unmixed substances, or they can he mixed manually by the user. Software presents the user with a summary of the interactions that have been identified at each stage, and a software user-interface guides further testing

Claims

CLAIMSWhat I claim is: 1. A method of investigating the chemical affinity between one or more target substances and a set of one or more test substances comprising the steps of 1) placing a solution of said target substance or substances in a non-flowing chamber of a flow-cell that is separated froni a flow-through chamber by a semipermeable or pOrOUS membrane, 2) moving a carrier fluid Ihat carries discrete batches of one or more test substances in solution through said how-through chamber, 3) detecting with a detector the retardation and spreading of said test substances that results from their chemical interactions with said target substance.
2. A method as claimed in claim 1 where said target substance is a niacromolecule.
3. A method as claimed in claim I where said larget subsiance is a biological material.
4. A method as claimed in claim 1 where said target substance is a protein or peptide.
5. A method as claimed in claim 1 where said target substance is a nucleic acid.
6. A method as claimed in claini I where said larget subsiance is a carbohydrate. polysaccharide.lipid, complex, non-biological synthetic compound, naturally occurring compound. virus.microorganism or synthetic macromolecule.
7. A method as claimed in any previous claim where said test suhsances are small organic molecules, inorganic molecules. ligands. drugs, fragments for drug discovery, organic compounds. peptides, proteins. nucleotides, nucleosides, nucleic acids, oligonucleotides, DNA, RNA, coenzynies, cofactors, signaling compounds, agonists, antagonists, inverse agonists, hormones, vitamins, lipids, inhibilors or dyes.
8. A method as claimed in any previous claim where the tinie taken for one or more test subsiances to reach a de1ecor is measured, where a longer time indicates a stronger interaction between the test substance in question and the target substance or substances.
9. A method as claimed in any previous claim where the width of peaks of test substances are measured with a detector, where a wider width indicates a stronger interactions between the test substance in question and the target substance or substances.
10. A method as claimed in any previous claim where the temperature of the flow-cell or the carrier fluid is controlled.
II. A method as claimed in any previous claim where the eflect ol varying the temperature on the passage of test substances is measured.
12. A method as claimed in claim 11 where the temperature of the flow-cell or the carrier fluid is controlled, where one or more test substances become bound strongly or irreversibly to the target substance, where the temperature is raised, and where the temperature at which said bound substance is released is measured.
13. A method as claimed in any previous claim where said rest substances are passed through said tlow-cell one by one.
14. A method as claimed in claims 1 to 12 where said test substances are passed through said flow-cell in batches containing two or more test substances. a
15. A method as claimed in claim 14 where each test substance is tested at least once in combination with a set of one or more other test substances focussing on variations in the allinily of the rest substance under consideraUon, where a reduction in affinity in [lie presence of another test substance indicates competitive binding by both test substances to a single site on the target substance, while binding that is undiminished in the presence of another test substance (that also binds) indicates independent binding to separate sites.
16. A method as claimed in claim 15 in which several test substances are seleded that are known to have strong affinity to the target substance hut which bind independently irom each other.and said selected substances are combined with all remaining members of a set of test substances in order to classify said remaining members in terms of their binding properties.
17. A method as claimed in claim 16 in which computer software and automatic fluid handling equipment including a sampler are used to classify as described said members automatically.
18. A method as claimed in any previous claim where two tlow-cells are used in parallel where the first flow-cell contains a target substance in its non-tiowing chamber, and where the second flow-cell is essentially identical in every respect except that is does not contain the target suhsance.
19. A method as claimed in claim 18. where the behaviour of test eotiipounds that pass through said first flow-cell is compared with their behaviour when they pass through said second flow-cell, where differences in behaviour correspond to interactions between the test compounds in question and the target substance.
20. A method as claimed in any previous claim where said target substance is dissolved in a liquid but the carrier fluid is a gas containing the test substances in a gaseous forni.
21. An apparatus for carrying out Ihe method of any previous claim, which includes a flow-cell that possesses 1) a non-flowing chamber that has an inlet and an outlet and is in fluid communication with one side of a porous or semi-permeable membrane, and 2) a flow-through chamber that also has an inlet and an outlet and is in fluid communication with the other side of said membrane.
22. An apparatus as claimed in claim 21 including a means of introducing fluids containing test subsiances into the inlet of said [low-through chamber, and a detector that connected lo the oullet of said flow-through chamber.
23. An apparatus as claimed in any previous apparatus claim where said flow-through and non-tlowing chambers are long conduits that are in eonmiunieation with each other through said membrane along at least part of their lengths.
24. An apparatus as claimed in claim 23 where said tiow-through and non-flowing chambers have one or more bends that increase their lengths while keeping their inlets and outlets relatively close to each other.
25. An apparatus as claimed in any previous apparatus claim where said chambers or conduits are formed by making indentations or depressions in the surfaces of two blocks that are subsequently mated such that said membrane is sandwiched between them.
26. An apparatus as claimed in claim 25 where said blocks arc made from metal, glass, ceramic, concrete, plastic or a composite material.
27. An apparatus as claimed in claim 26 where said flow-through and non-flowing chambers are mirror images that mate with each other giving communication between the chambers through said membrane along their lengths.
28. An apparatus as claimed in any previous apparatus claim where the temperature in the flow-cell can he controlled and set to a predetermined value.
29. An apparatus as claimed in any previous apparatus claim where the temperature in the flow-cell can be controlled and varied at will or according to a predetermined schedule.
30. An apparatus as claimed in any previous apparatus claim which possesses Iwo or more identical flow-cells that are connected to at least one detector.
31. An apparatus as claimed in claim 30 where a plurality of flow-cells are connected to a single deleclor using a switching valve.
32. An apparatus as claimed in any previous apparatus claim where a set or subset of samples of test suhstances can he loaded automatically from an array of samples in containers by a sampling device that is placed upstream of said how-cell allowing said samples to he introduced into said how-cell.