Patent Application
20120132572
The present invention relates to compositions comprising spherical beads which are encoded with radiofrequency codes through incorporation of a radiofrequency chip. The beads are preferably made of polymeric material and can for example be used for the synthesis of chemical libraries, e.g. solid-phase chemical libraries. Preferred materials include some that minimise interference in biochemical assays (fouling). The invention also relates to batch and continuous methods of producing such compositions, including emulsion-polymerisation methods. In a further aspect, the present invention relates to an apparatus for analysing radiofrequency-encoded beads. Also provided are methods for detecting and/or analysing and/or sorting beads, as well as methods for processing beads once they have been analysed and/or sorted.
A number of prior art documents describe the use of radiofrequency chips for the encoding of beads to be used for the synthesis of chemical libraries.
U.S. Pat. No. 6,265,219 (Chiron) describes transponder tagging of constituents used in compound synthesis. The synthesis members comprise a crown for solid synthesis and a stem in which the transponder is incorporated. Polyethylene, Teflon and fluorinated polymers are specifically proposed as materials for the synthesis members. The synthesis members are irregularly shaped and not suitable for high-throughput handling when dispersed in fluid compositions.
U.S. Pat. No. 6,319,668 (Discovery Partners International) describes matrices with memories, i.e. tagging of matrix materials with identifying information. Use of the matrices in the production of combinatorial libraries is described. The described tagging methods include radiofrequency tagging. Manual or automated coating of memory devices and casting or dipping is proposed, but the document does not provide methods that ensure the synthesis of spherical beads. Furthermore, no process for production of hydrophilic beads containing radiofrequency tags is provided.
U.S. Pat. No. 6,087,186 (Irori) describes labeled combinatorial synthesis libraries and methods and apparatuses for labelling individual library members of a combinatorial synthesis library with unique identification tags. Radiofrequency tags are amongst the proposed tags. Coating or encasing of tags is described, but the processes do not ensure formation of spherical beads.
WO 98/46548 (Zeneca) describes radiofrequency encoded chemical library synthesis particles which comprise a read-only radiofrequency tag linked to a solid phase. Their use in biological screening methods is also described. A process of ‘dicing’ beads out of a wafer is described. A spherical shape of the beads is proposed, but no process for its preparation is provided. The tags used are fairly large. Furthermore, only a limited number of hydrophobic polymeric materials are proposed for the bead. Some of these materials may cause inappropriate fouling in biological systems.
In conclusion, the prior art describes complex laborious methods for producing radiofrequency encoded beads, including dipping and dicing and manual insertion of beads. The beads are often large and irregularly shaped which makes them unsuitable for many high-throughput applications. Furthermore, many of the polymeric materials that are provided in the prior art only have a limited applicability for biological screening methods, as they result in fouling.
Radiofrequency chips and their antenna must be quite small if they are to be inserted into synthesis particles for use in high throughput screening. Such particles are typically spherical beads with a diameter less than 2 mm. Hence the size of the chip including antenna must be less than 2 mm long. Such small antennas typically operate at 2.45 GHz, which is a standard frequency for small radiofrequency identification systems.
Thus, there is a need for improved compositions comprising a plurality of beads that are optimally shaped for high throughput applications. Furthermore, there is a need for smaller radiofrequency-labeled beads and for novel non-fouling bead materials. There is also a need for methods of producing such compositions. In particular, there is a need for methods that allow efficient production of a large number of beads, and methods that allow to incorporate radiofrequency chips operating at a frequency of about 2.45 GHz into bead materials that are not readily compatible with the radiofrequency chip surface.
When large numbers of polymer beads, such as more than 10,000 beads, are to be analysed one by one at an acceptable total analysis time, the beads are typically dispersed in a liquid and passed through a measuring section of the analysis instrument.
WO 2005/062018 A2 discloses an apparatus for sorting and analysing beads.
The present invention is in one aspect directed to a bead analyser/sorter comprising a rotatable, circular capture body comprising a plurality of through-going inlets and antennae for radiofrequency identification, which is particularly suited for analysing beads each having incorporated therein a radiofrequency chip operating at a frequency of about 2.45 GHz.
The present invention is also directed to various methods.
Rønnekleiv et al. (submission at the 2005 IEEE Int. Ultrasonics Symp., Sept. 18-21, 2005, Rotterdam, The Netherlands) disclose the design of micromachined resonators for fish Identification.
In a further main aspect, the present invention relates to spherical encoded beads comprising an ultrasound identification chip.
In a first main aspect, the invention relates to a composition comprising a plurality of spherical beads, wherein a radiofrequency chip operating at a frequency of in the range of 2.2-2.7 GHz is embedded within each of said beads and wherein essentially each of said beads is individually identifiable on the basis of radiofrequency identification. Preferably, said beads comprise polymeric material comprising hydrophilic and/or hydrophobic moieties.
In a further main aspect, the invention relates to a method for the production of a composition comprising a plurality of spherical radiofrequency-identifiable polymeric beads comprising the steps of:
In a further main aspect, the invention relate to a method for the production of a composition comprising a plurality of spherical radiofrequency-identifiable polymeric beads comprising the steps of:
Radiofrequency encoding of beads through incorporation of radiofrequency chips provides a number of advantages over other means of encoding chips, including ease of detection and ease to avoid mix up between beads. Application on large scale has previously been hampered due to the large size of the radiofrequency chips (resulting in large beads), due to problems of producing large numbers of beads and due to problems with handling irregularly shaped beads. The problems have been solved in the present invention wherein methods are provided for large-scale batch or continuous preparation of spherical beads.
The spherical shape of beads makes it easy to handle the beads in high-throughput systems, in particular the high-throughput apparatuses of the present invention described herein. The spherical shape of the beads furthermore ensures optimal mechanical bead strength, because no protrusions can break off and mechanical stress is maximally distributed over the bead. A spherical shape of beads also minimises risk of beads sticking to solid surfaces or to each other. In particular sticking of multiple beads to each other is important to avoid in order to allow individual analysis of beads. Thus, spherical shape ensures a maximum ease of handling beads without breaking beads, and further enables handling of single beads.
For some uses of the compositions of the invention, hydrophilic beads are more suitable than hydrophobic beads. This can be the case when compositions of the invention are used in biological screening assays. Hydrophilic materials, in particular PEG-based polymers, generally interfere much less in such assays than hydrophobic materials. Radiofrequency chips do normally not have hydrophilic surfaces and can thus not be readily incorporated into a bead made of hydrophilic core material. In the present invention, this problem has been solved by providing a coating of the chips that allows the chip to disperse into hydrophilic droplet of an emulsion containing hydrophilic monomers to be polymerised. Thus, in some embodiments of the present invention, the radiofrequency chip is, prior to incorporation into a bead, coated with a layer (an ‘interphase’) that renders it hydrophilic and the chip will end up embedded within the bead through an interphase consisting of different material than the core.
Also incorporation of radiofrequency chips into hydrophobic bead material can be problematic due to unsuitable surface characteristics of the radiofrequency chips. The present invention also solves such problems through suitable coating of the chips prior to embedding.
The present invention relates in a further aspect to an apparatus for analysing radiofrequency-encoded beads. The apparatus is highly suitable for the analysis of beads of the composition of the present invention. The apparatus in one embodiment comprises a rotatable, capture body, such as a circular disc comprising a plurality of through-going inlets, wherein an individual bead from a composition comprising different beads can be fixed to the disc at the end-position of a through-going inlet by applying a pressure drop over said disc comprising said through-going inlets, The pressure drop results in beads being sucked (i.e, detachably fixed) onto the surface of the planar disc on top of the through-going inlets. As essentially all of the bead is present on top of the disc (i.e. extends from the surface rather than being contained in a recess therein), analysis of bead properties can be performed more readily and more easily while still ensuring a high through-put rate. When the capture body, preferably in the form of a planar disc comprising a plurality of through-going holes, is rotated, the beads are transferred from the position where they initially became attached to the disc to fixed positions wherein suitable devices for radiofrequency identification and/or analysing and/or sorting the beads can be operated in order to e.g. detect and/or analyse and/or sort at least one bead of a plurality of beads.
An important advantage of the apparatus of the present invention is that it allows stepwise analysis of the composition of beads. This allows to prolong the reading time which is a significant advantage e.g. if there is a poor signal-to-noise ratio. Furthermore, the apparatuses of the invention allow radiofrequency identification at small distance from the bead. This is also important if the signal-to-noise ratio is poor.
The present invention offers several solutions to the problems associated with prior art proposals for achieving an efficient sorting of e.g. radiofrequency-encoded beads:
The present invention ensures, when compared to the prior art,
In summary, when compared to fluid dynamics based instruments, the present invention allows for measurement exposure times orders of magnitude higher while maintaining comparable through-put rates, i.e. total number of beads screened per total screening time.
In one aspect of the present invention there is provided an apparatus for analysing a plurality of spherical radiofrequency-identifiable beads, said apparatus comprising
In another aspect of the present invention there is provided an apparatus for analysing a plurality of spherical radiofrequency-identifiable beads, said apparatus comprising
In still another aspect of the present invention there is provided an apparatus for analysing a plurality of spherical radiofrequency-identifiable beads, said apparatus comprising
The apparatus in one embodiment further comprises an analysing device for analysing results being generated from the detection of the radiofrequency code of beads, wherein said analysis enables individual beads to be characterised and/or identified and optionally also sorted.
Also provided in accordance with the present invention are methods for detecting and/or analysing and/or sorting beads, as well as methods for processing beads once they have been analysed and/or sorted. In one aspect the methods comprise the steps of diverting the beads to the bead sorting apparatus of the invention, detecting the radiofrequency code of at least one bead, and sorting at least one bead based on an analysis of the measurement result.
When data for the identification of all encoded beads in the bead population have initially been recorded on a data storage medium, i.e. data for the identification of all encoded beads in the bead population have been recorded prior to the actual step of identifying individual beads, all radiofrequency codes will already have been stored on the data storage medium. The total set of radiofrequency codes is therefore available and can thus be used for analysing and/or identifying individual encoded beads. An individual encoded bead will be identified once a match is found between the recorded radiofrequency code for the bead to be identified and the already stored radiofrequency codes for all beads.
Definitions
spherical—is used to indicate an essentially spherical shape of a bead of the invention. This is includes moderate deviations from a perfect spherical shape, e.g. moderately spheroidal shapes such as prolates or oblates, having an aspect ratio of between 0.8 and 1.2,
surface—part of a bead that is in contact with the surrounding medium.
interphase—layer between the radiofrequency chip and the core of the bead.
core—part of the bead that is not in contact with the surrounding medium. When an interphase is present, the core is furthermore not in contact with the radiofrequency chip.
hydrophilic—a composition that forms a contact angle with water of less than 60°, preferably less than 30°.
matrix—cross-linked polymer material.
radiofrequency chip—device that can communicate with an antenna through radiofrequency electromagnetic waves.
radiofrequency identification—identification on the basis of radiofrequency electromagnetic waves. essentially each of said beads—in connection with a specified criterion, this indicates that more than 90%, such as more than 95%, e.g, more than 98%, such as more than 99%, e.g. more than 99,5%, such as more than 99.9%, e.g. more than 99.99%, such as more than 99.999% of the beads fulfill the specified criterion.
emulsion, dispersion, suspension—liquid system comprising two immiscible liquids one of said liquids forming droplets in a continuum of the other of said liquids, said droplets being stabilised in said continuum by the combined action of stirring and the presence of a compound situated at the interface between the two phases.
fouling—indicates adhesion to biomolecules, such as DNA molecules, DNA-analog-molecules, RNA-molecules, RNA-analog-molecules, amino-acids, proteins, enzymes, lipids, antigens, viruses, cells, and the like.
surfactant—compound that stabilises droplets in an emulsion.
PEGA—PEG-acrylamide copolymer (may be alkylated on amide).
POEPOP—Polyethyleneglycol-polyoxypropylene copolymer.
SPOCC—Polymer obtained by ring opening polymerisation of partially or fully 3-methyloxetan-3-ylmethyl alkylated PEG.
HYDRA—PEG-tri-aminoethylamine star copolymer.
swelling—when beads are capable of swelling, any physical measurement, including size determinations and volume determinations, refer to measurements conducted for the swelled bead.
Compositions of the Invention
In a main aspect, the invention relates to a composition comprising a plurality of spherical beads, wherein a radiofrequency chip is embedded within each of said beads and wherein essentially each of said beads is individually identifiable on the basis of radiofrequency identification.
Compositions of the invention preferably comprise more than 103 of said beads, such as more than 104 of said beads, e.g. more than 105 of said beads, such as more than 106 of said beads, e.g. more than 107 of said beads, such as more than 108 of said beads, e.g. more than 109 of said beads, such as more than 1010 of said beads.
Beads of the composition are preferably suitable for solid-phase synthesis. Thus, preferably, essentially each bead comprises at least one site for chemical functionalisation to which a ligand or a bioactive species can be attached. Bioactive when used herein indicates causing a measurable change in a system comprising biomolecules. Biomolecules may for example be DNA molecules, DNA-analog-molecules, RNA-molecules, RNA-analog-molecules, amino-acids, proteins, enzymes, lipids, antigens, viruses, cells and the like. Preferably, the site for chemical functionalisation comprises a reactive group or a scaffold comprising two or more reactive groups.
Shape and Size of Beads
Compositions of the invention comprise beads that are spherical. Spherical shapes can be characterised by their aspect ratio (=diameter/height). Spherical shapes of the invention have an aspect ratio of between 0.8 and 1.2, most preferably between 0.9 and 1.1. ‘Spherical’ is not intended to indicate that the surface of the bead is entirely smooth, minor surface irregularities may occur. In spherical beads of the invention, the distance from the gravitational centre to any point on the surface of the bead is preferably in the range of from a quarter of the average distance from the gravitational centre to the surface to less than four times the average distance from the gravitational centre to the surface. Preferably, this distance is in the range from half of the average distance to less than two times. More preferably, this distance to any point of the surface is in the range from 0.75 to 1.25 times the average distance from the gravitational centre to the surface. The size of beads of the invention may vary, but preferably, essentially each of said beads has a volume of between 0.04 mm3 and 4 mm3, preferably between 0.1 mm3 and 0.5 mm3.
Radiofrequency Chips
Essentially each of the beads of the composition of the invention is individually identifiable, i.e. can be discriminated from other beads of the invention on the basis of its radiofrequency code contained within the radiofrequency chip. Radiofrequency chips used in the beads of the compositions of the invention are small. Preferably, a radiofrequency chip is smaller than 1 mm×1 mm×1 mm, more preferably smaller than 0.5 mm×0.5 mm×0.5 mm.
Radiofrequency chips can have a read-only memory, or, alternatively, a read-and-write memory. The chips preferably comprise an internal antenna. Preferred are furthermore chips wherein the individual identification is based on an identification number of 16, 32, 64 or 128 bits. Suitable chips can for example 0.4 mm×0,4 mm RFID p-chips with embedded antenna, supplied by Hitachi. Other suitable chips include ELAMS™ chips from Biomedic Data Systems and TIRIS™ chips from Texas Instruments. In one embodiment of the present invention the chips comprise a spherical antenna, whereby it is obtained that the reading of the chip becomes insensitive to the orientation of the radiofrequency waves.
Highly preferred chips are those operating at a frequency of in the range of 2.2-2,7 GHz, e.g. around 2.45 GHz.
Bead Material
Typically, the beads of the composition of the invention comprise polymeric material.
The spherical beads of the composition of the invention are characterised by a number of features. One essential feature is the presence of a radiofrequency chip operating at a frequency of in the range of 2.2-2.7 GHz which is embedded within the bead material. Beads that contain an only partially embedded chip will often be suitable for most applications, but preferably the chip is entirely embedded within the bead, i.e. fully surrounded by bead material. Thus, preferred embodiments of composition of the invention are ones in which in essentially all beads of the composition more than 50% of the surface of the radio-frequency chip is embedded within the bead, preferably more than 75%, more preferably more than 90%, most preferably essentially 100%, most preferably 100%. Beads of the present invention are normally micro-porous, whereby it is obtained that compounds can diffuse through the bead material. However, they normally do not contain large cavities, and thus are normally not vessels or vials.
The bead material surrounding the chip consists of a core, a surface and optionally an interphase. The surface is the part of the bead that is in contact with the surrounding medium, usually a liquid. The core is the part of the bead, usually the bulk part of the total bead material, that is not in contact with the surrounding medium. If there is no interphase, then the core is in contact with the chip. However, in a number of embodiments, there is an interphase between the core and the chip, composed of different material than the core. Such an interphase may be necessary to allow incorporation of the chip into the bead material.
Core and Surface Materials
Any material capable of forming spherical beads is in principle suitable for use in the production of beads of the invention. Preferably, the core material of a bead is polymeric. In some embodiments, the core comprises or consists of hydrophilic polymeric material. In other embodiments, the core comprises or consists of hydrophobic polymeric material. In some embodiments, the surface of the beads comprises or consists of the same material as the core. In other embodiments, the surface consists of other material than the core. For instance, beads of the fatter type can be obtained by chemical surface-modification of beads after polymerisation. Such surface-modification may include chemical functionalisation to render the beads suitable for solid-phase synthesis.
In some important embodiments, the surface of said beads comprises hydrophilic moieties. In particular, more than 50% of the surface, such as more than 60%, e.g. more than 70%, such as more than 80%, e.g. more than 90%, such as more than 95%, e.g. more than 99% of the surface of each of said beads consists of said hydrophilic moieties. As mentioned above, the core of said beads preferably comprises hydrophilic moieties. Preferred examples of such hydrophilic moieties are polyethylene glycol moieties, preferably cross-linked polyethylene glycol moieties, or polyamine moieties, or polyvinylamine moieties, or polyol moieties.
In other embodiments, the surface of said beads comprises hydrophobic moieties.
The choice of the type of beads, hydrophilic or hydrophobic, surface-modified or not, may depend on the application of the beads that is envisaged. A number of criteria may play a role, including physical characteristics, e.g. swelling, compatibility with aqueous solutions, compatibility with the type of molecules, such as biomolecules, to be attached, bioassays in which the beads are going to be screened, etc.
Encoded polymer beads according to the invention can be prepared from a variety of polymerisable monomers, including styrenes, acrylates and unsaturated chlorides, esters, acetates, amides and alcohols, including, but not limited to, polystyrene (including high density polystyrene latexes such as brominated polystyrene), polymethylmethacrylate and other polyacrylic acids, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidenechloride and polydivinylbenzene. In other embodiments, the beads are prepared from styrene monomers or PEG based macro-monomers. The polymer is in preferred embodiments selected from the group consisting of polyethers, polyvinyls, polyacrylates, polymethacrylates, polyacylamides, polyurethanes, polyacrylamides, polystyrenes, poiycarbonates, polyesters, polyamides, and combinations thereof. Highly preferred surface and core moieties include cross-linked PEG moieties, polyamine moieties, polyvinylamine moieties, and polyol moieties.
Preferred hydrophilic materials include PEG-grafted resins and PEG-cross-linked resins. PEG-grafted resins, such as TentaGel S (Rapp, W. In Combinatorial Peptide and Nonpeptide Libraries: A Handbook; Jung, G., Ed.; John Wiley & Sons, 1998, pp. 425-464), and PEG-cross-linked resins, such as PEGA (polyethylene glycol-polyacrylamide copolymer) (Renil et al (1998). 3. Pept. Sci. 4: 195-210); POEPOP (polyoxyethylene-polyoxypropylene) (Renil and Meldal (1996) Tetrahedron Lett. 37: 6185-6188); POE-PS3 (polyoxyethylene-polystyrene) (Buchardt and Meldal (1998) Tetrahedron Lett. 39: 8695-8698); SPOCC (Rademann et al.
(1999). 3. Amer. Chem. Soc. 121: 5459-5466) and HYDRA (Groth et al. (2000) J. Chem. Soc., Perkin Trans. 1:4258-4264) are aqueous compatible, and e.g. in general suitable for high-resolution MAS-NMR analysis, PEGA supports have proven useful for enzymology studies, for example, the screening of peptide or peptide-based inhibitor libraries (Smith and Bradley (1999)3. Combi. Chem. 1: 326-332; St. Hilaire, et al (1999) 3. Combi, Chem, 1: 509-523; Rosse et al. (2000) J. Combi. Chem. 2000, 2: 461-466).
In some preferred embodiments, the bead comprise a polymer selected from the group consisting of SPOCC, PEGA, HYDRA, POEPOP, PEG-polyacrylate copolymers, polyether-polyamine copolymers, cross-linked polyethylene diamines.
In a highly preferred embodiment, beads of the compositions of the invention comprise cross-linked polymeric material (i.e. a matrix) selected from the group consisting of polyoxetane-triethyleneglycol, polyoxetane-tetraethyleneglycol, and polyoxetane-pentaethylene-glycol, including any combination and/or derivative thereof. The matrix preferably comprises the structure:
wherein n is a number between 2 and 600, such as between 2 and 100, for example between 2 and 20, such as between 2 and 10. In one preferred embodiment, n is 2 or 3.
Another highly preferred matrix is selected from the group consisting of polyglycerol-triethyleneglycol, polyglycerol-tetraethyleneglycol, and polyglycerol-pentaethylene-glycol, including any combination and/or derivative thereof. The matrix preferably comprises the structure:
wherein n is a number between 2 and 600, such as between 2 and 100, for example between 2 and 20, such as between 2 and 10. In one preferred embodiment, n is 2 or 3,
Yet another highly preferred matrix is selected from the group consisting of poly(acryl)amide-triethyleneglycol, poly(acryl)amide-tetraethyleneglycol, and poly(acryl)amide-pentaethyleneglycol, including any combination and/or derivative thereof. The matrix preferably comprises the structure:
Irrespective of whether n is 1, 2, or 3, it is preferred in one embodiment that R is —CONH2. In another embodiment, R is —CONMe2. In a further embodiment, R is —CO2Me, and in a still further embodiment, R is —CN. Irrespective of whether n is 1, 2, or 3, and irrespective of whether R is —CONH2, —CONMe2, —CO2Me, or —CN, X can be —O— or —NH—. A more preferred matrix is one wherein n is 2, wherein R is —CONH2, and wherein X is —O—.
The above types of polymeric materials can be prepared as described in PCT/DK02/00687 and references therein.
Poly(alkylene)glycol Based Amino Polymers
Other suitable polymer matrices include those illustrated as formula 3 below, which can be prepared by exhaustive reduction of amide groups in the matrix of formula 4.
wherein ñ is a real number and designates the average degree of polymerisation (dp) of poly(alkylene)glycol in the range of from 3 to 2000.
The above types of polymeric materials can be prepared as described in PCT/DK2004/000461 and references therein.
Poly(aminoalkylene) Polymer Materials
Other suitable polymer materials for the beads of the compositions of the invention include cross-linked, optionally substituted, poly(aminoalkylene), of the formula I:
wherein A is a cross linking unit of functionality ≧2,
Other suitable materials include a beaded and cross-linked poly(aminoalkylene) matrix obtained by radical polymerisation of a molecule of formula IV having a radical reactive group R4R″CR″CY
wherein n is a number from 0 to 10;
wherein m is a number from 3 to 15,000;
wherein o is number 0 or 1;
wherein p is a number >0 and <m;
wherein Y is a heteroatom
wherein R″, R′″, R4, and/or R5 are hydrogen or optionally substituted saturated or unsaturated alkyl or optionally substituted aryl groups,
Methods for generating the above-mentioned beaded and cross-linked matrices include radical polymerisation methods. When the polymer matrices are made by radical polymerisation methods, there is further provided in accordance with the present invention a polymer matrix comprising a plurality of substituted amino groups, wherein the polymer matrix is obtained by a radical polymerisation method in combination with the further step of converting—after the polymerisation step—at least some of the amino groups to functional groups NR6R7, of formula V:
wherein R6 and R7 independently are H or an organic group formed by reaction of the amino groups of the polymer matrix according to the invention with an alkylating or acylating agent.
Polyethyleneimine Polymers
Further suitable matrix materials to be used in beads of compositions of the invention include cross-linked polymer matrices formed from a macromonomer comprising a polyethyleneimine functionalised with at least one fragment comprising a vinyl group, wherein said fragment can be polymerised using radical or ionic initiators to form the cross-linked polymer matrix.
The polyethyleneimine and the vinyl group can be linked by a unit Z preferably selected from a carbonyl group, a sulfone group, an aryl group, and derivatives thereof. The cross-linked polymer matrix preferably comprises the structure:
wherein ñ is an integer in the range of from 5 to 1500 and ã and õ are integers in the range of from 0 to ñ.
In further preferred polymers, the unit Z can be selected from the group consisting of CO; CO—(CH2)m; SO2; CS; and CNH; C6H4; and C6H4—CO—(CH2)m, where 0≦m≦10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In another preferred embodiment there is provided a polymer matrix comprising the structure:
wherein ñ is an integer in the range of from 2 to 1550, and
wherein the sum of x and y is an integer of more than 0 and at the most ñ−1.
The above types of polymeric materials can be prepared as described In PCT/Dk2004/000330 and references therein.
PD-DVB
A preferred hydrophobic polymer to be used for production of beads of the composition of the Invention is PS-DVB (polystyrene divinylbenzene). PS-DVB has been widely used for solid-phase peptide synthesis (SPPS), and has more recently demonstrated utility for the polymer-supported preparation of particular organic molecules (Adams et al. (1998) J.Org.Chem. 63:3706-3716). When prepared properly (Grøtli et al. (2000) J.Combi.Chem.2:108-119), PS-DVB supports display excellent properties for chemical synthesis such as high loading, reasonable swelling in organic solvents and physical stability.
Interphase Materials
Beads of the compositions of the present invention in some embodiments comprise an interphase for attaching the core bead material to the radiofrequency chip. Thus, in such embodiments, the radiofrequency chip is embedded within the bead through an interphase. Said interphase preferably comprises less than 10% of the total volume of the bead (excluding the chip), preferably less than 5% of the total volume of the bead. Interphase material can e.g. be coated onto the radiofrequency chips by plasma deposition and/or chemical vapour deposition and/or UV grafting and/or etching and/or silane grafting.
In a preferred embodiment, the interphase comprises an inner coating for protecting the chip, and an outer coating for binding the coated chip to the bead material. The inner coating is, in such embodiments, in contact with the chip, whereas the outer coating is in contact with the core material of the bead.
Suitable protective inner coatings can for Instance comprise or consist of one or more of the following materials: glass, organic polymers, ceramics, plastics, such as polyoxymethylene, polyethylene, polycarbonate, polystyrene, epoxy resin, PEEK, and fluorinated polymers, such as e.g. Teflon. In particular, the inner coating comprises inorganic glass or organic polymer.
The outer coating of the interphase, if present, has the function of binding and/or making the coated chip compatible with the polymeric material of the bead. This can be achieved in a number of ways. In some embodiments, the outer coating comprises a monomer which will be integrated into the polymeric material of the core of the bead by polymerisation. In other embodiments, the outer coating comprises material (a ‘compatibiliser’) which makes the chip compatible with polymeric material without becoming part of the polymer, i.e. it ensures that the coated chip can disperse and is miscible with the material to be polymerised. In some embodiments, the outer coating may contain, in addition to the compatibiliser, an initiator or co-initiator for initiation of polymerisation.
The following materials are presently preferred as interphase materials:
Silane Materials Suitable for Glass Surface Treatments:
acrylic-silane (monomer), peroxide-silane (initiator), photoinitiator-silane (photo-initiator), polyethyleneglycol-silane (compatibiliser), aminosilane (co-initiator), e.g. 4,N,N-di-methyl-methylsilane-aniline, N,N-diethyl(trimethylsilylmethyl)amine, (N,N-dimethylaminopropyl)-trimethoxysilane, (3-diethylaminopropyl)trimethoxysilane, or tri-methoxysilane-bound polyethyleneimine polymers.
Materials Suitable for Liquid Phase UV Grafting on Plastics:
acrylic-anthraquinone (monomer), peroxide- anthraquinone (initiator), polyethyleneglycol-anthraquinone (compatibiliser), amino anthraquinone (co-initiator), e.g. 4,N,N-di-methyl-methyl-anthraquinone-aniline, N,N-diethyl(trimethyl-anthraquinonemethyl)amine, (N,N-dimethylamino-propyl)trimethoxyanthraquinone, (3-diethylaminopropyl)trimethoxy-anthraquinone.
Gas Plasma Deposited Materials:
Outer coating materials can also be deposited by gas plasma deposition: Particularly useful for the present application are low-intensity alternating current plasma deposition techniques, such as low-intensity radiofrequency-pulsed plasma deposition techniques or low-intensity sub-radiofrequency plasma deposition techniques, such as low-intensity 50 Hz plasma deposition. Preferred materials to be deposited by gas plasma deposition include acrylic acid, ethanol, acetonitrile, acrylonitrile, acryloylchloride, ethylamine, acetonitrile, and tetraglyme.
Both the inner coating and the outer coating are only optional parts of the interphase (which itself also only is optional), Thus, it is also envisaged that an interphase only consists of the inner coating materials described above and does not contain outer coating materials. Conversely, an interphase can consist of outer coating materials and not contain inner coating material.
Methods for Producing Beads and Compositions of the Invention
The invention also relates to methods of producing compositions comprising a plurality of spherical radiofrequency-identifiable polymeric beads, such as the compositions according to the invention as described herein.
In a first main aspect, the invention relates to a batch emulsion-polymerisation method for the production of a composition comprising a plurality of spherical radiofrequency-identifiable polymeric beads comprising the steps of:
Initiation of polymerisation may be obtained in different ways. In some embodiments, the initiator is added independently of the chips, either after or upon addition of the chips to the emulsion. In other embodiments, the radiofrequency chips are coated with an initiator, thus ensuring that polymerisation only occurs around the chips.
Often, a combination of an initiator and a co-initiator will be used. In preferred embodiments, the initiator may be added during any of the steps i) to iv) of the above method, and the addition of the co-initiator or its activation is timed such that polymerisation does not occur before formation of droplets containing radiofrequency chips. In one such embodiment, exemplified in Example 5 herein, an initiator is added to the emulsion before introduction of the chips, and a co-initiator is added after introduction of the chips. In another embodiment, exemplified in Example 8, the chips are coated with an initiator, and a co-initiator is added after mixture of the first liquid, the second liquid and the radiofrequency chips.
The steps in the above batch preparation may furthermore include heating of the emulsion, bobbling with argon to remove oxygen, or other procedures known in the art that promote emulsion polymerisation. Furthermore, the resulting product may be washed and/or filtered to obtain a composition suitable for further use.
In a further main aspect, the invention relates to a method for continuous production of a composition comprising a plurality of spherical radiofrequency-identifiable polymeric beads comprising the steps of:
The radiofrequency chips are in the above method capable of dispersing into the second stream of monomeric material.
Initiation of polymerisation may be obtained in different ways in the above continuous production method. In some embodiments, the initiator is added independently of the chips, either before, after or upon addition of the chips to the emulsion.
In one embodiment, the addition of an initiator of polymerisation occurs after formation of droplets,
In a preferred embodiment, said addition of an initiator of polymerisation is performed Immediately prior to entry of said second stream into said first stream such that substantially no polymerisation occurs before formation of the droplets.
In other embodiments, the radiofrequency chips are coated with an initiator, thus ensuring that polymerisation only occurs around the chips. In this latter embodiment, it is important that said coated radiofrequency chips are added immediately prior the formation of droplets such that substantially no polymerisation occurs before formation of the droplets.
Often, a combination of an initiator and a co-initiator will be used. For example, an initiator of polymerisation may be provided in the second stream and a co-initiator is provided in the first stream, such that initiation of polymerisation starts upon contact between said first and second stream. In one such embodiment, exemplified in Example 9 and 10, the chips are coated with an initiator, and a co-initiator is added after mixture of the first liquid, second liquid and radiofrequency chips.
The steps in the above continuous preparation process may furthermore include heating of the emulsion, bobbling with argon to remove oxygen, or other procedures known in the art that promote emulsion polymerisation. Furthermore, the resulting product may be washed and/or filtered to obtain a composition suitable for further use.
In some embodiments of the above batch and continuous methods, the initiator and/or co-initiator is/are added in an inactive form and polymerisation is initiated by activation of the initiator, e.g. by heat or UV light.
In some embodiments of the above batch and continuous methods of the invention, the radiofrequency chips that are provided have been coated with material, e.g. a compatibilizer, that has rendered their surface more hydrophilic and the said second liquid or second stream comprises hydrophilic monomers to be polymerised. In other embodiments, the radiofrequency chips that are provided have been coated with a material, e.g. a compatibilizer, that has rendered their surface more hydrophobic and said second liquid or second stream comprises hydrophobic monomers to be polymerised. Thus, the radiofrequency chips that are used in the above batch and continuous methods can e.g. be coated with any of the materials described in the ‘Interphase materials section’ above, including monomers, compatibilisers and combinations thereof.
The above batch and continuous methods of the invention may optionally be followed by a step of separating beads that have one radiofrequency chip from beads that have no radiofrequency chip or more than one radiofrequency chip. Such separation may e.g. be done flow or using a bead sorter, e.g. as described in Examples 6 and 7.
Surfactants
In preferred embodiments of the present invention, a surfactant is included in the polymerisation process. Suitable surfactants include for example anionic surfactants, such as the following anionic surfactants (supplied by Huntsman):
Alkyl Benzene Sulphonate Derivatives: NANSA series
Alpha Olefin Sulphonates: NANSA LSS series
Alcohol Sulphates: EMPICOL series
Alcohol Ethoxy Sulphates: ALKANATE W, EMPICOL series
Alkylphenol Ethoxy Sulphates: ALKANATE W, EMPICOL series
Naphthalene Sulphonate Derivatives: DEHSCOFIX series
Sulphosuccinates: EMPICOL S, SURFONIC DOS series
Phosphate Ester Derivatives: ALKANATE P, EMPIPHOS, SURFONIC PE series
Sodium Vinyl Sulphonate: HARTOMER 4900
Examples of suitable non-ionic surfactants include the following (supplied by Huntsman):
Alkylphenol Alkoxylates: EMPILAN NP, EMPILAN OP, SURFONIC N, TERIC N, TERIC X series
Alcohol Alkoxylates: EMPILAN K, SURFONIC L, TERIC A series
EO/PO Copolymers: EMPILAN PF, SURFONIC POA, TERIC PE series
Alkyl Polysaccharides: ALKADET, ECOTERIC series
Alkylamine Ethoxylates: EMPILAN AMO, EMPILAN AMT, SURFONIC T, TERIC M series
Other alternative suitable surfactants include alcohol alcoxylates, such as BEROL EP 25 and BEROL EP 35, supplied by Akzo Nobel, and Nonyl Phenol Ethoxylates, such as BEROL 02 or BEROL 09 (also supplied by Akzo Nobel).
A further alternative suitable surfactant includes sorbitain monolaurate.
In view of the above-mentioned methods for the preparation of beads, it should be understood that the invention also relates to a composition comprising a plurality of radio-frequency-identifiable polymeric beads obtainable by the methods described above.
Apparatuses of the Invention
The present invention in a further main aspect provides an apparatus comprising devices for bead manipulation and bead detection. This apparatus is highly suitable for manipulation and detection of beads of the compositions of the invention.
In one aspect of the present invention there is provided an apparatus for analysing a plurality of spherical radiofrequency-identifiable beads, said apparatus comprising
In one embodiment, the apparatus has a ratio R between a) the average diameter of the beads being manipulated, and b) the diameter of the through-going inlets, R=a/b, is more than 2, such as more than 4, for example more than 6, such as more than 8, for example more than 10, such as more than 12, for example more than 14, such as more than 16, for example more than 18, such as more than 20, for example more than 25, such as more than 30, for example more than 35, such as more than 40, for example more than 50, and preferably less than 100.
Preferably, the capture body of the apparatus is a planar disc.
The bead manipulation device preferably comprises a mechanical bead handling apparatus comprising a vacuum container comprising a capture disc for bead capture and manipulation.
Once captured onto the capture disc of the vacuum container the radiofrequency code of the beads can be detected, e.g. by using an antenna. The generated data can be stored on a data storage medium and analysed. Accordingly, the apparatus can further comprise a device for bead analysis based on the data generated by the detection. In an even further embodiment the apparatus can also comprise a device for bead processing based on the data generated by the detection device and/or the data generated by the analysing device.
The principle of the operation of the apparatus according to the present invention is illustrated in
The circular capture body (103) can be manipulated, such as rotated in a step-wise fashion, so that a bead, once it has been firmly fixed onto an inlet, can be transferred to a detection section (106) where the radiofrequency code of the bead can be measured by a suitable device (107). The detection section and the detection device will preferably be stationary, whereas the step-wise motion of the capture disc will transfer—in a step-wise fashion—beads to the detection section, one bead after the other. Accordingly, the term “section” as used herein will be understood to refer to a particular volume that contains the full track of the capture holes or parts thereof, and through which at least one capture hole can be manipulated. By the “track of the capture holes” is meant the spatial geometry described by the moving capture holes. In the case of a rotating capture disc with capture holes arranged along a circle centred around the axis of rotation of the capture disc, the track of the capture holes is a circle. In preferred embodiments of the present invention a section contains only parts of the track of the capture holes, and preferably all capture holes can be manipulated through a section. In cases where an apparatus of the present invention includes a guiding plate, a section typically is fixed relative to the guiding plate. In the preferred cases, where the guiding plate comprises a guiding channel, a section typically refers to a volume including a specific part of the guiding channel. Accordingly, any bead, which is detected by the detection device is positioned in a detection section. The beads are preferably detected in stationary mode, i.e. in between the step-wise motions, which are required in order to rotate the capture disc and transfer beads from one section (e.g. a loading section) to another section (e.g. a detection section).
It is to be understood that the capture body can be of various geometries other than circular and can be manipulated in various ways other than rotation. As an example the capture body can be spherical with capture holes arranged along a circle and can be rotated around and axis perpendicular to the geometrical plane of the capture holes and going through the centre of the circle described by the capture holes. As a further example a capture body can comprise a rectangular capture surface with a rectangular array of capture holes arranged in rows and columns, and can e.g. be manipulated in directions parallel to the rows and columns of the capture holes. Furthermore the loading and/or unloading can be performed batch wise. As an example, a capture body can be loaded with beads by immersing the capture surface of the capture body into a dispersion of beads, and can be unloaded by immersing the capture surface in dispersion liquid and disconnecting the vacuum body from the vacuum and optionally connecting the vacuum container to a pressurised source of dispersion liquid.
Examples of suitable detection devices are radiofrequency detection devices, such as radiofrequency detection devices comprising a radiofrequency antenna connected to a radiofrequency antenna controller. One example of a radiofrequency antenna is a radiofrequency antenna comprising a cylindrical ferrite core circumvented by a copper coil.
In one preferred embodiment of the present invention the radiofrequency detection device and the RFID chip operate at a frequency of in the range of 2.2-2.7 GHz, such as around 2.45 GHz.
In another aspect of the present invention there is provided an apparatus for analysing a plurality of spherical radiofrequency-identifiable beads, said apparatus comprising
In this variant, the capture disc comprise radio-frequency antenna positioned at every capture hole of said capture disc, thus, the radio-frequency antennas rotate together with the capture disc. As an example the radiofrequency antenna comprise a thin layer of microstructured electrically conductive material in the shape of e.g. a spiral around each capture hole. Such antenna can be manufactured by standard deposition and etching methods known from the electronics industry. In this way the distance between the radiofrequency chip and the antenna for reading the chip is determined by the distance from the radiofrequency chip to the surface of the radiofrequency encoded bead.
Preferably, the detection devices within the above variant further comprise one or more radiofrequency antenna oriented at various angles to the surface of the capture disc.
Optionally one or more antenna are positioned at the back of the vacuum container and focussed on the detection section of the apparatus. When using radiofrequency chips, which can not be read from any direction, the combined operation of antenna placed at the front and at the back of the vacuum container ensure that all radiofrequency encoded beads can be read regardless of orientation.
Examples of suitable analysing devices are e.g. computers with computer programs installed for comparison of a radiofrequency code with stored radiofrequency codes. Suitable computer programs can furthermore e.g. generate a sorting result on the basis of the comparison, keep track of the position of beads and their associated sorting results, and control an actuator of an optional sorting section of an apparatus of the present invention. Furthermore, computer programs can control the means for treating a bead in one or more optional treating sections of the present invention, such as keeping track of synthesis beads and their individual predetermined building block exposure sequences and control the addition of specific building blocks to said one or more optional treating sections in accordance thereto.
Once a bead has been subjected to a detection step and optionally also a analysis step, the capture body can be further manipulated, such as rotated, preferably in the same orientation as previously, so that a bead having been detected and optionally also analysed, subsequently enters a processing section (108) where the bead can optionally be processed by a suitable processing device (109).
A processing or treatment step can include a chemical reaction. By chemical reaction is meant a process that breaks existing chemical bonds, such as covalent bonds, ionic bonds, or hydrogen bonds, and forms new chemical bonds within a given chemical compound. As an example chemical compounds with a desired structure can be synthesised on beads for solid phase synthesis, so-called synthesis beads, by exposing the beads to building blocks in a specified sequence. As an example the treating device can comprise a plurality of compartments comprising chemical building blocks said compartments being connected to the treating section by at least one tube for diverting individual chemical building blocks to the treating section, such that specific beads can be exposed to specific building blocks in the treating section.
In a similar and still further step of operating the capture body, the bead can be transported to an unloading section (110) by suitable manipulation of the capture body. The bead in question can be removed from the capture body e.g. by reversing the pressure drop having been exerted during the aforementioned steps, i.e. by reversing - once the bead enters the unloading section - the pressure drop P2′-P1′ over the inlet to which the bead has been attached during the above-mentioned operations.
The bead sorting and detection apparatus described in principle above allows a fast and reliable sorting, detection and identification of a plurality of polymer beads. Using the bead sorting apparatus disclosed herein it is possible to perform methods enabling a detection in a single hour of as many as more than 10,000 beads, such as more than 20,000 beads, such as at least 30,000 beads, such as at least 36,000 beads, such as at least 76,000 beads per hour, with detection times of about 1/20 second or less. Detection times in this range are orders of magnitudes longer than the approximately 10 microseconds allowed for by a purely fluid dynamics based systems.
Furthermore, the sensor for detecting the coming of a bead, which is an essential component of the purely fluid dynamics based system, is rendered superfluous by the present invention due to the accurate mechanical control of the capture body being operated by a stepper motor.
The vacuum container comprises a first (outer) surface onto which beads can be captured, and through-going inlets extending from the first surface to a second (inner) surface. The captured beads can be transferred between different “utility sections”—defined by fixed positions occupied by different beads over time—when the vacuum container is rotated around a central axis. By “utility sections” is meant sections such as e.g. loading sections, detection sections, analysing sections, processing sections, and unloading sections.
The beads can finally be removed from the capture vacuum container at an unloading section (110) by rotating the capture vacuum container so that a bead on the first surface is contacted by a bead stopper (202) which forces the bead away from the first surface of the capture vacuum container. The use of a bead stopper eliminates the need for supplying a strong vacuum near the first surface of the capture vacuum container at the unloading section.
In one aspect of the invention there is provided a vacuum container comprising a) a circular capture disc comprising a plurality of through-going inlets, b) a circular capture disc support supporting the capture disc at a distal end thereof (at the perimeter) and being connected at a proximal end (at the central axis) of the capture disc to c) a hollow shaft, wherein the hollow shaft is preferably fitted with a shaft hole so that a vacuum (i.e. a pressure below 1 bar) can be applied to the interior of the vacuum container, and d) a stepper motor operably linked to a momentum transfer split for transferring the momentum from the stepper motor to the vacuum container, thereby causing the vacuum container to rotate in a controlled, step-wise fashion.
In the above description the pressure drop over the capture disc is generated by applying a vacuum to the interior of the vacuum body. It is to be understood though that the pressure drop can be generated in other ways. One alternative is to apply a pressure to the dispersion liquid contacting the outer surface of the capture disc by e.g. connecting a pressurised dispersion liquid reservoir thereto. A further alternative is to apply a vacuum to the inside of the vacuum container and at the same time to apply a pressure to the dispersion liquid on the outside.
The above-described vacuum container comprising a rotatable capture disc for bead sorting is preferably integrated into an apparatus for bead sorting further comprising the following features
An overview of the apparatus for bead sorting according to the invention is described herein below with reference to
Overview of Bead Sorting Apparatus
The following paragraphs describe with reference to
It is to be understood that the beads can be suspended in any sufficiently easy flowing liquid. By easy flowing is meant that the liquid has a not too high viscosity, such as a viscosity less than 10000 milli Pa s, such as less than 1000 milli Pa s, preferably less than 100 milli Pa s, e.g. between 0.5 milli Pa s and 10 milli Pa s, such as about 1 milli Pa s, which is the viscosity of water at room temperature. As an example aqueous solutions of minerals and/or biological material can be used as suspension liquid.
In one preferred embodiment of the invention the suspension liquid has little or practically no absorbance of electromagnetic radiation at the frequency used to read the RFID tag inside the bead, whereby it is it obtained that the operating radiofrequency radiation of the RFID-chip of the bead is not damped by the dispersion liquid.
In a further preferred embodiment of the invention the suspension liquid has little or practically no absorbance of electromagnetic radiation at 2,45 GHz, e.g. an organic liquid, such as a hydrocarbon such as hexane, a halogenated solvent such as di-chloromethane, or a mineral oil such as Isopar-M supplied by ExxonMobil Chemical.
In yet a further preferred embodiment of the present invention the suspension liquid is water and the operating frequency of the RFID-chip is about 2.45 GHz. In this embodiment the suspension liquid does in fact absorb the electromagnetic radiation at the operating frequency of the RFID-chip. To minimize the undesired absorbance of the electromagnetic radiation by the suspension liquid, the suspension liquid is removed from the detection section and optionally from a further part of the guiding channel, said part of the guiding channel including the detection section.
Hence, in still another aspect of the present invention there is provided an apparatus for analysing a plurality of spherical radiofrequency-identifiable beads, said apparatus comprising
This embodiment is illustrated in
The water reservoir (304) diverts aqueous liquid such as water to the water feeding section of the apparatus. The water in the water reservoir preferably has a free surface for ensuring a water pressure inside the guiding channel of approximately 1 bar.
The first water pump (305) is connected to the vacuum connecting piece of the apparatus for maintaining a vacuum inside the vacuum container. The vacuum ensures that beads remain firmly fixed to the capture disc of the vacuum container during transfer to a detection section. The transfer occurs when beads having been fixed to the capture disc of the vacuum container by the applied vacuum pressure are rotated in step-wise motions by the action of a stepper motor operated by a computer (307).
The radiofrequency code of beads are detected by a detection device (303) and optionally also stored on a data storage medium in a computer (307).
In one preferred embodiment of the present invention the operating radiofrequency code of the beads and the detection device is 2.45 GHz.
The computer (307), or a set of different computers, can be used for controlling the stepper motor, as well as controlling the detection device, storing data, analysing the data obtained, and controlling the piston valve at the unloading section.
A third water pump (310) generates a vacuum for removing beads from the capture disc at a sorting section. The removal of beads is ensured by the actions of a piston valve (308) connecting the vacuum of the third water pump to the sorting section. A second filter (309) can retain beads removed at the sorting section. A second water pump (311) generates a vacuum for removing beads not removed at the unloading section from the capture disc at the unloading section.
Operation of the Bead Sorting Apparatus
The below sections describe the actions routinely performed when operating the above-described apparatus.
Initially, the computer (307) and the detection device are turned on. The valve (308) is closed and the water reservoir (304) is filled with demineralised water. The first water pump (304) is activated, whereby a pressure of less than 0.5 bar is maintained inside the vacuum container. The third water pump (310) is started whereby a pressure of 0.1 bar is maintained downstream from the second bead filter (309), The second water pump (311) is started whereby a pressure of 0.1 bar is maintained downstream from the first bead filter (312).
A computer program is run which controls the stepper motor (313), the valve (308), and the detection device, so that the vacuum body is rotated in a step-wise fashion and so that radiofrequency codes are obtained, stored, and analysed in the computer every time a capture hole is momentarily at rest in the detection section. The valve (308) is controlled on the basis of the result of the analysis of the codes, thereby enabling sorting of the beads.
Vacuum Container
A detailed illustration of one preferred embodiment of a vacuum container and a vacuum container housing is provided in
Capture Disc Holder of Vacuum Container
A cylindrical capture disc holder (415) makes it possible to apply a pressure drop over the disc. The capture disc holder (415) can have an outer diameter of e.g. 100 mm. The capture disc holder can support the disc at the perimeter of the disc while the central section of the capture disc holder is supported by a hollow shaft (410), preferably a hollow stainless steel shaft, through which shaft one can apply a first vacuum of less than e.g. 0.5 bar, The hollow stainless steel shaft (410) can have an outer diameter of about 6 mm and an inner diameter of about 4 mm.
Vacuum Container Housing
The vacuum container housing according to this embodiment serves the purposes of containing the dispersion liquid, holding the vacuum container in place, connecting the inside of the vacuum container to a vacuum, and transferring rotational momentum to the vacuum container.
The vacuum container housing in one embodiment preferably comprises:
A suitable means for stepwise rotating the disc is a stepper motor with e.g. 200 steps per round mounted on the shaft and arranged so that the motor causes the disc and the container to rotate around a common central axis. In this way, the capture holes are moved along a planar, circular path. The stepper motor comprises an electronic stepper motor controller for controlling the motion of the stepper motor.
The guiding plate preferably comprises a circular guiding channel, such as e.g. a 1 mm deep circular guiding channel having an outer diameter of e.g. about 81,5 mm and an inner diameter of e.g. about 78.5 mm carved therein, said guiding plate further comprising a number of through-going inlets for supplying and retracting beads, or more preferably dispersion liquid comprising beads, to and from the guiding channel of the guiding plate, wherein the guiding plate is optionally attached to a guiding plate holder (401) for holding the guiding plate.
The vacuum container and vacuum container housing can be constructed in different ways in order to serve the purpose of transferring beads from one section of utility to another. The construction is not critical as long as it permits the container to function according to the principles of the invention. In
The individual components of a vacuum container comprise a) a circular capture disc comprising a plurality of a through-going inlets forming a circle close to the perimeter of the capture disc, b) a circular capture disc support supporting the capture disc at a distal end and being connected at a proximal end to c) a hollow shaft preferably fitted with a shaft hole so that a vacuum can be applied to the interior of the vacuum container, and of a vacuum container housing comprising d) a stepper motor operably linked to a momentum transfer split for transferring the momentum from the stepper motor to the vacuum container thereby causing the vacuum container to rotate in a controlled step-wise fashion, and optionally further components, is disclosed in the following.
The capture body (416) can comprise e.g. a 100 mm diameter and 5 mm thick plastic disc comprising two planar, circular sides, a first and a second side. The capture holes (105) can comprise any suitable number of through-going inlets, such as e.g. about 100 cylindrical through-going inlets of varying diameter, the diameter being e.g. about 0.2 mm at the first side of the disc (to which the beads are attached), and the diameter being e.g. about 2.0 mm at the second side of the disc. In a preferred embodiment the number steps of the stepper motor is divisible by the number of capture holes, and the capture holes are equidistantly spaced, whereby it is obtained that all capture holes are at rest at the exact same positions.
The capture holes (through-going inlets) can e.g. be arranged along an 80 mm diameter circle positioned about 10 mm from the perimeter of the disc.
Utility Sections of the Apparatus for Bead Sorting
Loading Section
The loading section (104) can comprise a volume of e.g. at least 1 mm3, said volume being confined in an essentially cylindrical space extending from the surface of the first side of the disc and into the dispersion liquid and positioned at the circle described by the capture holes.
In a preferred embodiment the loading section comprises an elongated volume extending along the track of the capture holes for a distance corresponding to several times the average distance between neighbouring capture holes, such that at all times during the operation of the apparatus multiple capture holes, such as at least ten capture holes, are contained in the loading section, whereby the probability of a capture hole capturing a bead while traversing the loading section is increased compared to the case of a loading section containing only one capture hole at a time. In order to further increase the chance of capture holes capturing a bead inside the loading section the number of mobile beads in the loading section should be maximised. By “mobile beads” is meant beads that can be captured by a an empty passing capture hole by action of the flow of dispersion liquid towards the capture hole. At low numbers of beads (the bead number) the number of mobile beads (the mobile bead number) increases with increasing bead number up to a certain critical bead number, the clogging bead number, where beads become immobilised by friction and adhesion interactions with neighbouring beads and with the solid surfaces surrounding the loading section. Hence, the bead number in the loading section should be kept just below the clogging bead number. It should be noticed then, that the clogging bead number depends on various parameters, such as e.g. bead size, bead composition, dispersion liquid composition, dispersion liquid flow velocity, and loading section geometry. To avoid the bead capture rate from becoming the throughput limiting factor in the operation of the apparatus the mobile bead number should be maximised.
As already mentioned, the mobile bead number can be increased by increasing the clogging bead number. As an example this can be achieved by inducing static flow in the loading section, e.g. by stirring, such as stirring with a magnetic stirrer, or by infusing dispersion liquid at one end of the loading section and withdrawing dispersion liquid from the distal end of the loading section. In many cases a more pronounced effect can be achieved by inducing an alternating flow field.
A further obvious way of increasing the mobile bead number is to extend the loading section along the track of the capture holes. Obviously the length of the loading section is limited by the total length of the track of the moving capture holes. In the case of a circular capture hole track the total length of the capture hole track can be increased by increasing the diameter of the capture hole track. In cases where the capture holes are arranged along a circle in a capture disc the total length of the capture hole track can be increased by increasing the diameter of the capture disc. If for instance a 500 mm capture disc be used, a total of up to 500 capture holes can be arranged along a circle with 3 mm between neighbouring capture holes. The loading section can then be designed such that at least 400 capture holes be present in the loading section at all times during operation. This significantly increases the probability of a capture hole capturing a bead in the loading section compared to the case of a 100 mm diameter capture disc with the same inter-capture hole distance.
The unloading section (110) preferably also comprises an at least 1 mm3 essentially cylindrical space extending from the surface of the first side of the disc and into the dispersion liquid and positioned at the circle described by the capture holes at e.g. 180° from the loading section.
Detection, Analysis and Unloading Sections
The detection section can be defined by a 1 mm3 spherical space extending from the surface of the first side of the disc and into the dispersion liquid. The detection section (106) for detecting radiofrequency codes, and optionally also analysing the data resulting from the detection, is preferably positioned on the circle perimeter defined by the capture holes at an angle of 90° from both the loading section (104) and the unloading section.
Accordingly, beads are transferred from the loading section via the detection section to the unloading section. The detection section is so positioned that the stepwise rotation of the disc causes a through-going inlet (capture hole) to which a bead is fixed to be stationary (i.e. not moving) when the bead fixed to the through-going inlet passes the detection section. This ensures that individual beads can be detected in “stationary mode” during the movements generated by the stepper motor.
Once the beads have passed through the detection section (106), they are optionally transferred by further step-wise rotation to an analysis section (607).
Once the beads have passed through the detection section (106) and optionally an analysis section (607), they need to be unloaded from the capture disc. In the embodiment disclosed in
When a bead enters the unloading section it is firmly fixed onto a through-going inlet of the disc due to the pressure drop over the inlet. Any type of pressure controlling equipment can be used in an unloading section for normalising the vacuum or, preferably, for generating a reverse pressure drop over the part of the disc which at any one time is positioned in the unloading section. The pressure-controlling equipment can e.g. comprise a pipe for unloading beads, preferably of stainless steel, having a length of e.g. about 20 mm and an inner diameter of e.g. about 1.1 mm.
The pipe comprises a first end and a second end, and the pipe is preferably positioned perpendicular to the first side of capture disc, the first end of the pipe pointing towards the first side of the disc and being positioned about 1 mm from the capture disc and entering the unloading section, the second end of the pipe being connected to a second vacuum of 0.1 bar, whereby a reverse pressure drop, P1′-P2′=−0.4 bar, is generated over the capture disc at the unloading section,
The different sections of the bead sorting apparatus are illustrated in more detail in
With reference to
For example, the through-going capture inlet n will initially be located in the loading section for being loaded with a bead.
As the stepper motor rotates the disc a single step, the through-going capture inlet n will be rotated one step in the orientation of the rotation, At the same time, the through-going capture inlet n+1 will be located in the loading section for being loaded with a bead.
As the stepper motor rotates the disc another single step, the through-going capture inlet n+1 will be rotated one step in the orientation of the rotation. At the same time, the through-going capture inlet n+2 will be located in the loading section for being loaded with a bead, and so on.
As the stepper motor rotates the disc step-wise, the through-going capture inlet n will be rotated a plurality of steps in the orientation of the rotation. After a certain number of step-wise rotations, the through-going capture inlet n will have been rotated so many steps that it will be positioned in the detection section.
In the embodiment of the capture disc disclosed in
In the loading section (104) the beads are sucked onto the through-going capture inlets of the capture disc, and non-captured beads are removed in an excess bead unloading section (601).
There is also provided a liquid feeding section (605) in which e.g. water can be diverted to the guiding channel generating a flow of water in a direction away from the water feeding section. Also provided in this embodiment is an excess-bead flushing section (604) for flushing any non-captured beads away from the flushing section and towards the bead feeding section, whereby it is obtained that only captured beads proceed from the excess-bead flushing section towards the detection section. Examples of non-captured beads include non-captured beads sticking to captured beads, non-captured beads sticking to the surface of the capture disc, non-captured beads sticking to the walls of the guiding channel, and freely flowing non-captured beads. In this way it is ensured that non-captured beads do not pass the water feeding section.
Sorting Section
Once the beads have passed through the detection section (106) and an analysis section (408), they are optionally sorted into at least two fractions. In the embodiment disclosed in
Detailed disclosures of preferred embodiments of the sorting section are provided in
The sorting section is illustrated in a preferred embodiment in
In a further embodiment of the sorting section illustrated in
It is essential that the pressure drop over the vacuum outlet be sufficiently high for being able to draw away beads that have been blown from their capture holes from the guiding channel, yet sufficiently low to not remove captured beads from their capture holes.
In yet a further embodiment of the second, optional unloading section illustrated in
When both the unloading section and the sorting section are present the different sections are connected to different pumps or the same pump for generating a vacuum in the unloading section and sorting section, respectively.
Accordingly, using the apparatus for bead sorting disclosed herein above it is possible to perform a method wherein
Methods of the Invention
In a further main aspect, the invention relates to a method for detecting a radiofrequency code of at least one bead of a plurality of beads, said method comprising the steps of
In another main aspect, the invention relates to a method for identifying at least one bead of a plurality of beads, said method comprising the steps of
In a further main aspect, the invention relates to a method for sorting at least one bead of a plurality of beads, said method comprising the steps of
Each of the above methods, i.e. the method for detecting, the method for identifying and the method for sorting, can be part of a method for treating at least one bead of a plurality of beads, said method comprising carrying out the steps of one of the above methods and the further step of treating at least one bead of a plurality of beads, preferably based on the identification.
In a further main aspect, the invention relates to a method for treating at least one bead of a plurality of beads, such as polymer beads, said method comprising the steps of
In preferred embodiments of the above methods of the invention, the radiofrequency code of the at least one bead is detected within a time period of between 1 millisecond and 1 second, preferably between 10 milliseconds and 100 milliseconds, such as 50 milliseconds,
Furthermore, in preferred embodiments of the above methods of the invention, a total of more than 4000 beads is detected per hour, such as more than 5000 beads per hour, for example more than 10,000 beads per hour, such as more than 15,000 beads per hour, for example more than 20,000 beads per hour such as more than 25,000 beads per hour, for example more than 30,000 beads per hour such as more than 40,000 beads per hour, for example more than 50,000 beads per hour such as more than 60,000 beads per hour, for example more than 70,000 beads per hour, such as more than 80,000 beads per hour, for example more than 90,000 beads per hour such as more than 100,000 beads are detected per hour.
In further preferred embodiments of the above methods, the distance between the detection device and the at least one bead is between 0.1 mm and 5 mm, such as about 0.5 mm, during the detection.
In further preferred embodiments, the step of treating at least one bead of a plurality of beads comprises exposing the at least one bead to at least one chemical building block under reaction conditions suitable for the reaction of said building block.
A number of uses of the present invention are envisaged.
Thus, in a further main aspect, the invention relates to a method for recording individual reaction steps involved in the step-wise synthesis of a chemical compound on a radiofrequency-detectable bead, said method comprising the steps of
In another main aspect, the invention relates to a method for identifying a chemical compound having been synthesised on a radiofrequency-detectable bead, said method comprising the steps of
In a preferred embodiment, the assay is a binding assay performed by measuring the binding of a protein to a ligand bound to the bead,
In another preferred embodiment, the assay is performed by measuring an enzyme activity on a substrate bound to the bead.
In a further preferred embodiment, the assay is performed by measuring enzyme inhibition of a molecule bound to the bead.
In an even further preferred embodiment, the assay is performed by measuring receptor interaction with a compound bound to the bead.
In preferred embodiments of the above application methods, the bead is a bead from a composition of the invention as defined hereinabove,
Method for Deconvoluting a Conventional Library
In a further aspect, the invention provides a method for synthesising and deconvoluting a combinatorial library comprising the steps of:
(a) apportioning in a stochastic manner among a plurality of reaction vessels a plurality of beads on which a plurality of different compounds can be synthesised, wherein said plurality of beads comprises a population of detectably distinct beads each having a radiofrequency code, which distinctively identifies a respective bead before, during and after said synthesis from other beads,
(b) determining and recording the codes of said plurality of beads in order to track the movement of individual detectably distinct beads into particular reaction vessels of said plurality of reaction vessels, wherein said codes are determined prior to step (d);
(c) reacting the beads in each reaction vessel with a building block;
(d) pooling the beads from each reaction vessel;
(e) apportioning the beads in a stochastic manner among the plurality of reaction vessels;
(f) reacting the beads in each reaction vessel with another building block;
(g) recording the codes of said plurality of beads in order to track the movement of individual detectably distinct beads into particular reaction vessels of said plurality of reaction vessels, wherein said codes are recorded after step (e) and/or step (f);
(h) pooling the beads from each reaction vessel;
(i) iterating steps (e) through (h) as required in order to create a combinatorial compound library wherein member compounds of the library are associated with the detectably distinct beads and wherein codes of the detectably distinct beads are deconvolutable using tracking data provided by said recordal steps to identify the sequence of reactions experienced by the said detectably distinct beads.
The identification steps (step (c) and (d)) may be effected by use of any suitable method or apparatus for analysing the radiofrequency code of a bead.
Building Block Reactions
The beads of the compositions of the invention are applicable to any type of chemical reaction that can be carried out on a solid support. Such chemical reaction includes, for example:
1. 2+2 cycloadditions including trapping of butadiene;
2. [2+3] cycloadditions including synthesis of isoxazolines, furans and modified peptides;
3. acetal formation including immobilisation of diols, aldehydes and ketones;
4. aldol condensation including derivatisation of aldehydes, synthesis of propanediols;
5. benzoin condensation including derivatisation of aldehydes;
6. cyclocondensations including benzodiazepines and hydantoins, thiazolidines, -turn mimetics, porphyrins, phthalocyanines;
7. Dieckmann cyclisation including cyclisation of diesters;
8. Diels-Alder reaction including derivatisation of acrylic acid;
9. Electrophilic addition including addition of alcohols to alkenes;
10. Grignard reaction including derivatisation of aldehydes;
11. Heck reaction including synthesis of disubstituted alkenes;
12. Henry reaction including synthesis of nitrile oxides in situ (see 2+3 cycloaddition);
13. catalytic hydrogenation including synthesis of pheromones and peptides (hydrogenation of alkenes);
14. Michael reaction including synthesis of sulfanyl ketones, bicyclo] 2.2.2] octanes;
15. Mitsunobu reaction including synthesis of aryl ethers, peptidyl phosphonates and thioethers;
16. nucleophilic aromatic substitutions including synthesis of quinolones;
17. oxidation including synthesis of aldehydes and ketones;
18. Pausen-Khand cycloaddition including cyclisation of norbornadiene with pentynol;
19. photochemical cyclisation including synthesis of helicenes;
20. reactions with organo-metallic compounds including derivatisation of aldehydes and acyl chlorides;
21. reduction with complex hydrides and Sn compounds including reduction of carbonyl, carboxylic acids, esters and nitro groups;
22. Soai reaction including reduction of carboxyl groups;
23. Stille reactions including synthesis of biphenyl derivatives;
24. Stork reaction including synthesis of substituted cyclohexanones;
25. reductive amination including synthesis of quinolones;
26. Suzuki reaction including synthesis of phenylacetic acid derivatives; and
27. Wittig, Wittig-Horner reaction including reactions of aldehydes; pheromones and sulfanyl ketones.
Reference may also be made to Patel et al., (April 1996, DDT 1 (4): 134-144) who describe the manufacture or synthesis of N-substituted glycines, polycarbarnates, mercaptoacylprolines, diketopiperazines, HIV protease inhibitors, 1-3 diols, hydroxystilbenes, B-lactams, 1,4-benzodiazepine-2-5-diones, dihydropyridines and dihydropyrimidines. Reference may also be made to synthesis of polyketides as discussed, for example, in Rohr (1995, Angew. Int, Ed. Engl. 34: 881-884).
Chemical or enzymatic synthesis of the compound libraries of the present invention takes place on beads. Thus, those of skill in the art will appreciate that the materials used to construct the beads are limited primarily by their capacity for derivatisation to attach any of a number of chemically reactive groups and compatibility with the chemistry of compound synthesis. Except as otherwise noted, the chemically reactive groups with which such beads may be derivatised are those commonly used for solid state synthesis of the respective compound and thus will be well known to those skilled in the art. For example, these bead materials may be derivatised to contain functionalities or linkers including —NH2, —NHNH2, —ONH2, —COON, —SH, —SeH, —SO3H, —GeH, or —SiR2H groups.
It will also be appreciated that compounds prepared with the beads and/or process of the present invention may be screened for an activity of interest by methods well known in the art. For example, such screening can be effected by specialised flow cytometry invented from standard techniques such as described e.g. by Needels et al., (1993, Proc. Natl. Acad. Sci. USA 90: 10700-10704, incorporated herein by reference), Dower et al. (supra), and Kaye and Tracey (WO 97/15390, incorporated herein by reference).
Synthesis of a Combinatorial Compound Library
A combinatorial library in accordance with the present invention is a collection of multiple species of chemical compounds comprised of smaller subunits or monomers. Combinatorial libraries come in a variety of sizes, ranging from a few hundred to many hundreds of thousand different species of chemical compounds. There are also a variety of library types, including oligomeric and polymeric libraries comprised of compounds such as peptides, carbohydrates, oligonucleotides, and small organic molecules, etc. Such libraries have a variety of uses, such as immobilisation and chromatographic separation of chemical compounds, as well as uses for identifying and characterising ligands capable of binding an acceptor molecule or mediating a biological activity of interest.
The library compounds may comprise any type of molecule of any type of subunits or monomers, including small molecules and polymers wherein the monomers are chemically connected by any sort of chemical bond such as covalent, ionic, coordination, chelation bonding, etc., which those skilled in the art will recognise can be synthesised on a solid-phase support
Various techniques for synthesising libraries of compounds on solid-phase supports are known in the art. Solid-phase supports are typically polymeric objects with surfaces that are functionalised to bind with subunits or monomers to form the compounds of the library. Synthesis of one library typically involves a large number of solid-phase supports.
To make a combinatorial library, solid-phase supports are reacted with a one or more subunits of the compounds and with one or more numbers of reagents in a carefully controlled, predetermined sequence of chemical reactions. In other words, the library subunits are “grown” on the solid-phase supports. The larger the library, the greater the number of reactions required, complicating the task of keeping track of the chemical composition of the multiple species of compounds that make up the library. Thus, it is important to have methods and apparatuses which facilitate the efficient production of large numbers of chemical compounds, yet allow convenient tracking of the compounds over a number of reaction steps necessary to make the compounds.
Combinatorial libraries represent an important tool for the identification of e.g. small organic molecules that affect specific biological functions. Due to the interaction of the small molecules with particular biological targets and their ability to affect specific biological functions, they may also serve as candidates for the development of therapeutics.
Accordingly, small molecules can be useful as drug leads eventually resulting in the development of therapeutic agents.
Because it is difficult to predict which small molecules will interact with a biological target. intense efforts have been directed towards the generation of large numbers, or “libraries”, of small organic compounds. These libraries can then be linked to sensitive screens to identify the active molecules.
A number of libraries have been designed to mimic one or more features of natural peptides. Such peptidomimetic libraries include phthalimido libraries (WO 97/22594), thiophene libraries (WO 97/40034), benzodiazopene libraries (U.S. Pat. No. 5,288,514), libraries formed by the sequential reaction of dienes (WO 96/03424), thiazolidinone libraries, libraries of metathiazanones and their derivatives (U.S. Pat. No, 5,549,974), and azatide libraries (WO 97/35199) (for review of peptidomimetic technologies, see Gante, J., Angew. Chem. Int. Ed. Engl. 1994, 33, 1699-1720 and references cited therein).
The present invention also resides in a method of synthesising and deconvoluting a combinatorial library as described herein above. The codes of the plurality of beads are determined preferably before the first reaction step, although codes may be determined at any time before the first pooling step. Preferably, every time the plurality of beads is apportioned into reaction vessels, each one of the vessels is analysed to determine which of the detectably distinct beads are in each reaction vessel. A database of all the beads (or corresponding gridspaces, supra) can thus be updated to show the synthetic history of the compound synthesised on each bead.
During a reaction step, the beads in each reaction vessel are reacted with a building block required to assemble a particular compound. Assembly of compounds from many types of building blocks requires use of the appropriate coupling chemistry for a given set of building blocks. Any set of building blocks that can be attached to one another in a step-by-step fashion can serve as the building block set. The attachment may be mediated by chemical, enzymatic, or other means, or by a combination of these. The resulting compounds can be linear, cyclic, branched, or assume various other conformations as will be apparent to those skilled in the art. For example, techniques for solid state synthesis of polypeptides are described, for example, in Merrifield (1963, J. Amer. Chem. Soc. 35: 2149-2156). Peptide coupling chemistry is also described in “The Peptides”, Vol. 1, (eds. Gross, E., and J. Meienhofer), Academic Press, Orlando (1979), which is incorporated herein by reference.
To synthesise the compounds, a large number of the beads are apportioned among a number of reaction vessels. In each reaction, a different building block is coupled to the growing oligomer chain. The building blocks may be of any type that can be appropriately activated for chemical coupling, or any type that will be accepted for enzymatic coupling. Because the reactions may be contained in separate reaction vessels, even building blocks with different coupling chemistries can be used to assemble the oligomeric compounds (see, The Peptides, op. cit). The coupling time for some of the building block sets may be long. For this reason the preferred arrangement is one in which the building block reactions are carried out in parallel. After each coupling step, the beads on which are synthesised the oligomers or compounds of the library are pooled and mixed prior to re-allocation to the individual vessels for the next coupling step, This shuffling process produces beads with many oligomer sequence combinations. If each synthesis step has high coupling efficiency, substantially all the oligomers on a single bead will have the same sequence. That sequence is determined by the synthesis pathway (building block reactions and the order of reactions experienced by the beads) for any given bead. The maximum length of the oligomers may be about 50, preferably from 3 to 8 building blocks in length, and in some cases a length of 10 to 20 residues is preferred. Protective groups known to those skilled in the art may be used to prevent spurious coupling (see, The Peptides, op cit.).
With enough beads and efficient coupling it is possible to generate complete sets of certain oligomers, if desired. The appropriate size of the beads depends on (1) the number of oligomer synthesis sites desired; (2) the number of different compounds to be synthesised (and the number of beads bearing each oligomer that are needed for screening); (3) the effect of the size of the beads on the specific screening strategies e. g, fluorescence-activated cell sorters (FACS) to be used; and (4) the resolution of the encoding/detection methods employed.
Further Uses of the Invention
As described above a highly preferred use of the compositions of the invention is the use for the synthesis of a combinatorial chemistry library and/or a solid-phase combinatorial library.
Also relevant is the use of the combinatorial chemistry library defined above for the screening of bioactive compounds and/or drug discovery and/or affinity-ligand discovery.
The compositions may further be used in diagnostic method, e.g. as carriers for particular ligands or capture probes.
The compositions may also be used for animal tracking, e.g. by implanting a one or more beads of a composition into each of a plurality of animals so as to keep track of the animals. It should be understood that this is particularly useful for small animals.
Spherical Encoded Polymer Bead with Ultrasound Identification Chips
The current inventors have further developed certain polymer beads which are particularly useful for identification when suspended in aqueous liquids, such as water, The present inventors envisage that such polymer beads are particular useful in the processes described in detail above and in connection with the apparatus (possibly suitable modified to include an ultrasound transmitter and a corresponding ultrasound receiver) described above, or more generally the apparatus of the type disclosed in WO 2005/062018 A2.
Thus, the present invention also provides a spherical polymer bead comprising embedded therein an ultrasonic identification chip, said chip comprising one or more resonator cavities, the dimensions of each of the one or more resonator cavities giving rise to a ultrasonic resonance frequency of 20 kHz or more.
The spherical polymer bead can be identified by acoustic waves in the ultrasonic range, i.e. above 20 kHz. One advantage of acoustic waves for use in aqueous environments is the fact that acoustic waves propagate through water at very low losses. Especially the frequency range of 100-10,000 kHz is useful for identification of the above-described beads suspended in water.
The preparation of the ultrasonic identification chip can be accomplished according to the guidelines given in Rønnekleiv et al, (submission at the 2005 IEEE Int, Ultrasonics Symp., Sept. 18-21, 2005, Rotterdam, The Netherlands).
A large number of such microstructered chips can be manufactured by standard lithographic techniques known to persons skilled in the art. As an example, on the order of 100,000 quadratic chips with side length 0.5 mm can be made from one 200 mm diameter silicon wafer.
The preparation of the spherical beads can be accomplished as described hereinabove for the radiofrequency-encoded beads.
In a preferred embodiment of the present invention, the ultrasonic identification chip comprises a top coating layer (1901), a microstructured layer with at least one through-going hole, each forming the walls of a cavity (1902), and a sealing layer (1903), the top coating layer and the sealing layer forming respective end-walls of said cavities, cf.
In one embodiment of the present invention 13,104 ultrasonic chips are embedded inside spherical beads—one chip in each bead—by methods provided by the present invention. The beads are 1.5-3.0 mm in diameter. All the chips are squares with side length 1 mm. Each chip differs from the others by having a unique combination of number of cavities and cavity side lengths. The number of cavities range from 1 to 6, and independently the side cavity side lengths are chosen from six values in the range from 100-160 micrometers. The resulting set of chips have from 1 to 6 distinct resonance frequencies in the range from 200-600 kHz with each bead having a unique set of resonance frequencies.
In one embodiment of the invention, each of the one or more resonator cavities gives rise to a resonance frequency of in the range of 20-10,000 kHz, such as in the range of 100-10,000 kHz, in particular in the range of 500-8,000 kHz or in the range of 100-2,500 kHz.
The minimum size of the chips depends on the number of different codes needed and the frequency used for reading the code. The number of different codes possible, N, is given by,
where L is the number of different resonance frequencies detectable, and M is the maximum number of cavities on each chip.
An approximate correlation between the size of a cavity, d, and its resonance frequency, fr can be used,
f
r
=k
1
exp(−k2 d),
where k1 and k2 can be determined by fitting to experimental data. The data presented by Rønnekleiv et al. at the 2005 IEEE Int. Ultrasonics Symp., Sept. 18-21, 2005, Rotterdam, The Netherlands, is well approximated by k1=280 and k2=0.002 in the range 200-400 kHz. Inserting these values of k1 and k2 in the above expression and extrapolating to higher frequencies result in the following set of correlating fr and d values:
From these values it can be seen that a cavity of about 5 μm has a resonance frequency of in on the order of 2,000 kHz.
In one embodiment, of the present invention 125,673 ultrasonic chips are embedded inside spherical beads—one chip in each bead—by methods provided herein. The beads are 0.7-1.4 mm in diameter. All the chips are squares with side length 0.5 mm. Each chip differs from the others by having a unique combination of number of cavities and cavity side lengths. The number of cavities ranges from 1 to 9, and independently the side cavity side lengths are chosen from 9 values in the range from 60-100 μm. The resulting set of chips have from 1 to 9 distinct resonance frequencies in the range 100-1,000 kHz with each bead having a unique set of resonance frequencies.
In one embodiment of the present invention, which is illustrated in
In yet another embodiment of the present invention, about one million ultrasonic chips are embedded inside spherical beads—one chip in each bead—by methods provided by the present invention. The beads are 0.4-0.6 mm in diameter. All the chips are squares with side length 0.2 mm and thickness 0.1 mm. Each chip differs from the others by having a unique combination of number of cavities and cavity side lengths. The number of cavities ranges from 1 to 10, and independently the cavity side lengths are chosen from 10 values in the range from 5-30 μm. The resulting set of chips have from 1 to 10 distinct resonance frequencies in the range 1,000-5,000 kHz with each bead having a unique set of resonance frequencies.
The present invention also provides methods for reading ultrasonic encoded beads. In one preferred embodiment the bead sorting apparatus is equipped with at least one ultrasonic transmitter and at least one ultrasonic receiver. By positioning two or more pairs of transmitters and receivers at different positions along the guiding channel of the bead sorting apparatus, it is obtained that the ultrasonic chip can be read by at least one such transmitter-receiver pair regardless of the angular orientation of the ultrasonic chip.
The ultrasonic code of ultrasonic encoded bead is measured with the use of the bead sorting apparatus equipped with ultrasonic transmitter-receiver pairs according to the present invention. When an ultrasonic encoded bead is at rest in front of one of said ultrasonic transmitters in-between the step-wise rotation of the capture disc the ultrasonic transmitter transmits an ultrasonic sweep in the frequency range 100-1000 kHz. The corresponding ultrasonic receiver records the ultrasonic signal emitting from the ultrasonic encoded bead. The bead identity is derived from the set of signals recorded of each bead by the full set of receivers of the apparatus.
A bead analysis apparatus with auxiliaries for controlling the bead handling is constructed comprising (numbers referring
a rotating vacuum container comprising:
a vacuum container housing comprising:
a PMMA circular front plate comprising:
an unloading section comprising:
means for rotating the vacuum container comprising:
bead handling apparatus auxiliaries comprising:
means for reading the radiofrequency chip of the beads comprising
The bead sorting apparatus and auxiliaries described above are operated in the following way:
The first gear pump is started at 2500 rpm whereby a vacuum is generated inside the vacuum container whereby water is drawn from the guiding channel into the capture holes whereby the pressure inside the guiding channel is lowered and whereby water is drawn from the water reservoir into the water-feeding holes.
The second gear pump is started at 20% of maximum rotational speed whereby water is drawn from the unloading section of the guiding channel through the unloading bead container towards the second gear pump.
The pulse generator is started in continuous single pulse mode at 0.5 seconds between pulses and a pulse width of 0.5 milliseconds whereby the axis of the stepper motor is caused to rotate 3.6° every 0.5 seconds.
The bead feeding syringe is gently shaken in order to evenly disperse the beads in the water whereafter approximately 0.1 mL of the bead dispersion is infused into the guiding channel through the feeding hole.
The radiofrequency antenna are operated in automatic gain control mode, whereby the highest obtainable signal-to-noise ratio is obtained. The signals from the three antenna are sampled such that the triple measurements on each bead are sampled and identified on the basis of the three measurements.
The operation of the bead sorting apparatus described above results in a portion of the beads being transported from the bead feeding syringe to the unloading bead container, and in a sequence of identifications of radiofrequency-encoded beads.
Once the majority of capture holes have become occupied with beads the water level inside the bead sorting apparatus is lowered, such that the upper half of the capture surface is above water, whereby it is obtained that beads positioned inside the analysis section are surrounded by air instead of water. This is advantageous because air does not absorb the radiofrequency electromagnetic radiation used for identifying the beads.
The present method is easily up-scaled in terms of total number of beads measured by keeping the pulse generator running and by repeating the infusion of suspended beads at suitable time intervals such as every 140 seconds. Furthermore, the throughput of the current method can be increased by lowering the time between pulses generated by the pulse generator and reducing the time interval between infusion of suspended beads.
The bead handling apparatus and auxiliaries described in Example 1 were operated with the following operation parameters:
The first gear pump was running at 2500 rpm.
The second gear pump was running at 40% of maximum rotational speed.
The pulse generator was running in continuous single pulse mode with 0.25 seconds between pulses and a pulse width 0.5 milliseconds.
The bead feeding syringe was mounted on a syringe pump set to run in continuous withdrawal/infusion mode with volume setting 0.1 mL and rate setting 1.0 mL/min. It was noted that the actual volume of infused bead dispersion per withdrawal/infusion cycle was substantially less than the nominal value of 0.1 mL due to the combined mechanical bias of the syringe mounting and of the flexible plastic syringe itself.
The operation of the bead handling apparatus described above results in a portion of the beads being transported from the bead feeding syringe to the unloading bead container, and in a sequence of identifications of radiofrequency encoded beads.
The problem with more than one bead captured at a capture hole can be overcome by sorting away the more than one captured beads by methods described elsewhere in the present invention and run them through the apparatus a second time.
In order to develop novel ligands for use in chromatographic purification of proteins, a ligand library is prepared by the following method:
Compound Synthesis
200,000 PEGA-type polymer beads with diameters in the range 0.5-0.7 mm are subjected to a four step solid phase split-process-recombine combinatorial synthesis route involving ten different building blocks per step, whereby approximately 10,000 compounds, here ligands, are generated, each bead carrying one ligand, and each ligand being carried by 20 beads on the average.
In order to evaluate the affinity of the ligands towards a specific protein, the beads are exposed to an aqueous solution of a fluorescence labeled modification of the protein and subsequently weakly adhering fluorescence labeled protein is removed by washing. Now the beads that carry a ligand with high affinity towards the fluorescence labeled protein are strongly fluorescent, whereas beads carrying low affinity ligands are weakly fluorescent or non-fluorescent.
The bead handling apparatus with auxiliaries described in example 1 is modified in the following way: The radiofrequency antenna are replaced by an optical fibre connected at one end to a imaging window of the detection section at the 12.30 o'clock position of the front plate and at the other end to a photo-multiplier tube (PMT) equipped with a fluorescence emission filter for blocking the laser light and transmitting the fluorescence emission and further equipped with an electronic amplifier for amplifying the electronic output from the PMT and an A/D-converter for converting the analogues signal from the amplifier into a digital signal (measuring result).
The bead handling apparatus from example 1 is further equipped with a sorting section at the 10.30 o'clock position comprising a
The bead handling apparatus described in example 1 and the modified auxiliaries are operated in the same way as described in example 1 with the exception that instead of radiofrequency identifying the beads in the detection section, their total fluorescence is measured by the photo-multiplier tube. The measuring result is fed to a computer that generates an analysis result, in this case a sorting result, for each bead being measured by the following scheme: if the measuring result is greater than a pre-set value the sorting result=1, whereas, if the measuring result is less than or equal to the value, the sorting result=0.
Each bead, its associated sorting result, and its position on the capture disc from the detection section and forward is recorded by a computer.
At the sorting section each bead is removed from its capture hole, by briefly (50 milliseconds) opening the sorting valve, and transferred to the second bead filter (309) if its associated sorting result=1, whereas the bead is left on the capture disc if its analysis result=0.
At the unloading section all beads that were not removed at the sorting section are removed from the capture disc and transferred to the first bead filter (312),
In this way, two fractions of beads are generated, one fraction containing beads with a total fluorescence above the specified value, i.e. a fraction of beads carrying ligands with high affinity towards the fluorescence labeled protein and one fraction containing beads with a total fluorescence below the specified value, i.e. a fraction of beads carrying ligands with low affinity towards the fluorescence labeled protein.
Now, the chemical structure of the ligands carried by the fraction of strongly fluorescent beads can be analysed and determined to some degree of certainty by methods known by those skilled in the art. Prior art instrumentation and methods for total fluorescence based bead sorting exist, however, the performance of the apparatus and method of the present invention to our best knowledge supersedes prior art disclosures in terms of accuracy, i.e., fraction of correctly sorted beads, due to the precise spatial control of beads of the present invention.
The pre-set value involved in the generation of the sorting result must be sufficiently high for generating only a small fraction of beads with sorting result=1, such as 1% or less, such as 0.1% or less, whereby it is obtained that only beads carrying very high affinity ligands are separated. A proper pre-set value can be found by a trial and error method: After exposure to fluorescence labeled protein and washing a random fraction of the beads are run through the bead handling apparatus with a random pre-set value. If the fraction of beads with sorting result=1 is too low the experiment is repeated with a lower pre-set value, whereas, if the fraction of beads with sorting result=1 is too high the experiment is repeated with a higher pre-set value. This procedure is repeated until a proper pre-set value has been found.
The above method can also be used within drug discovery for synthesis and screening of drug candidates. In the case of drug discovery the compounds synthesised on the beads can be drug candidates, and can be screened against a relevant biological compound, such as e.g. an antibody.
The above method can further be used within catalyst development for synthesis and screening of catalyst candidates, in which case the compounds synthesised on the beads can be catalyst candidates, and can be screened against a relevant set of reactants.
Furthermore, the above method or the bead sorting by itself finds use within diagnostics, e.g. for screening biological fluids with regards to the presence of specific DNA or DNA-analogue (e.g. RNA, m-RNA, LNA) sequences. In the case of diagnostics the beads can carry single stranded DNA or DNA-analogue sequences, and can be screened against a biological compound comprising single stranded DNA or DNA-analogue sequences.
The method for ligand development described in example 3 involves the difficult step of determining the chemical structure of the high-affinity ligands. This step is by far the most time consuming, and often leads to ambiguous results. However, the need for this step can be eliminated by keeping track of each bead through its combinatorial synthesis route, i.e. its individual reaction vessel sequence, and after bead sorting, identifying the beads carrying high affinity ligands. In this way the chemical structures of the high affinity ligands are derived from the track of its host bead.
The method for compound synthesis described in example 3 is repeated but in this example with spatially encoded PEGA-type beads and with the added steps of
By this method the combinatorial synthesis route of each bead carrying a high affinity ligand is determined, on which basis the chemical structure of its ligand can be derived, whereby the undesired ligand analysis is avoided.
27 mL oil (Isopar M) is transferred to a glass reactor equipped with a stirring rod connected to a motor. The glass reactor is heated to 50° C. The oil is purged with argon in order to remove oxygen (O2). A monomer mixture is prepared by dissolving 0.8 g difunctional monomer (polyethyleneglycol-di-acrylamide, ca. 1900 g/mole), 0.8 g monomer (polyethyleneglycol-acrylamide, ca. 1900 g/mole), 0.08 g co-monomer (acrylamide), and 0.05 g surfactant (SORBITAIN MONOLAURATE) in 5 mL of demineralised water in a glass flask by stirring at room temperature for 30 minutes. While stirring, argon is bobbled through the monomer mixture in order to remove oxygen (O2). 0.07 g initiator (ammoniumperoxo-di-sulfate) is added to the monomer mixture, which is stirred for 5 more minutes. The stirring of the oil in the reactor is started by switching on the motor connected to the stirring rod. 1000 glass coated radiofrequency chips (ca. 0.5 mm×0.5 mm×0.2 mm) are added to the glass reactor. The monomer mixture is added to the glass reactor. 0.34 g co-initiator (tetra-methyl-ethyl-diamine) is added to the glass reactor. The mixture is stirred in the glass reactor at 50° C. for one hour.
The glass reactor is opened, the contents is poured into a filter funnel and washed in the funnel with solvents in the following order: dichloromethane, tetrahydrofuran, methanol, water. The resulting wet beads are sieved through a 0.7 mm mesh-size sieve and then through a 0.5 mm mesh-size sieve. The resulting 0.5-0.7 mm diameter fraction of beads is the product.
The product from example 5 is transferred to a column comprising a vertical glass cylinder equipped with a 0.3 mm mesh-size filter at the bottom. Demineralised water is poured upwards through the column at a low flow rate. The flow rate is carefully increased until the beads containing no radiofrequency chips start to float. These beads are removed from the top of the column. The flow rate is further increased whereby the beads containing one chip start to float. These beads (the product) are then removed from the top of the column.
The product from example 5 is separated into fractions of beads according to number of radiofrequency chips per bead with the use of a bead sorter (COPAS, Harvard Bioscience) on the basis of total light extinction. The fraction of beads with one radiofrequency chip is the product,
1000 glass coated radiofrequency chips (ca. 0.5 mm×0.5 mm×0.2 mm) are coated with polyethyleneglycol-silane and then immersed in a saturated aqueous solution of initiator (ammoniumperoxo-disulfate). The radlofrequency chips are removed from the solution and left to dry at room temperature.
27 mL oil (Isopar M) is transferred to a glass reactor equipped with a stirring rod connected to a motor. The glass reactor is heated to 50° C. The oil is purged with argon in order to remove oxygen (O2). A monomer mixture is prepared by dissolving 0.8 g difunctional monomer (polyethyleneglycol-di-acrylamide, ca. 1900 g/mole), 0.8 g monomer (polyethyleneglycol-acrylamide, ca. 1900 g/mole), 0.08 g co-monomer (acrylamide), and 0.05 g surfactant (SORBITAIN MONOLAURATE) in 5 mL of demineralised water in a glass flask by stirring at room temperature for 30 minutes, While stirring, argon is bobbled through the monomer mixture in order to remove oxygen (O2). The stirring of the oil in the reactor is started by switching on the motor connected to the stirring rod. The coated radiofrequency chips are added to the reactor. The monomer mixture is added to the glass reactor. 0.34 g co-initiator (tetra-methyl-ethyl-diamine) is added to the glass reactor. The mixture is stirred in the glass reactor at 50° C. for one hour.
The glass reactor is opened, and the product is washed as described in example 5.
1000 glass coated radiofrequency chips (ca. 0.5 mm×0.5 mm×0.2 mm) are coated with polyethyleneglycol-silane and then immersed in a saturated aqueous solution of initiator (ammoniumperoxo-disulfate). The radiofrequency chips are removed from the solution and left to dry at room temperature.
50 mL oil (Isopar M) is transferred to a glass flask and stirred with a magnetic stirring bar at room temperature. The oil is purged with argon in order to remove oxygen (O2). A monomer mixture is prepared by dissolving 0.8 g difunctional monomer (polyethyleneglycol-di-acrylamide, ca. 1900 g/mole), 0.8 g monomer (polyethyleneglycol-acrylamide, ca. 1900 g/mole), 0.08 g co-monomer (acrylamide), and 0.05 g surfactant (SORBITAIN MONOLAURATE) in 5 mL of demineralised water in a glass flask by stirring at room temperature for 30 minutes. While stirring, argon is bobbled through the monomer mixture in order to remove oxygen (O2). The coated radiofrequency chips are added to the monomer mixture, which is then transferred to a syringe pump. The oil is pumped from the glass flask with the use of a stepper motor to a first inlet of a first T-piece through a 1.0 mm inner diameter tube at a flow rate of 100 mL. per hour. The monomer mixture is pumped to the second inlet of the first T-piece at a flow rate of 10 mL per hour through a 0.3 mm inner diameter tube inserted in the exit tube from the T-piece such that the monomer mixture is infused into the oil stream 5 mm downstream from the T-piece at which point 1.0 mm diameter monomer droplets are generated inside the exit tube from the first T-piece. From the first T-piece the monomer-phase/oil-phase mixture is led to a first inlet of a second T-piece. A (10:1) mixture of oil and co-initiator (tetra-methyl-ethyl-diamine) is fed to the second inlet of the second T-piece. From the second T-piece the mixture is led through a 0.5 m long 1 mm inner diameter tube through an oil bath at 50 C where the monomer droplets polymerise. The resulting beads and the oil are gathered in a glass flask at 50° C. and stirred. The beads are washed as described in example 5.
1000 glass coated radiofrequency chips (ca. 0.5 mm×0.5 mm×0.2 mm) are coated with polyethyleneglycol-silane and then immersed in a saturated aqueous solution of initiator (ammoniumperoxo-disulfate). The radiofrequency chips are removed from the solution and left to dry at room temperature.
500 mL oil (Isopar M) is transferred to a glass flask and stirred with a magnetic stirring bar at room temperature, The oil is purged with argon in order to remove oxygen (O2). A monomer mixture is prepared by dissolving 0.8 g difunctional monomer (polyethyleneglycol-di-acrylamide, ca. 1900 g/mole), 0.8 g monomer (polyethyleneglycol-acrylamide, ca. 1900 g/mole), 0.08 g co-monomer (acrylamide), and 0.05 g surfactant (SORBITAIN MONOLAURATE) in 5 mL of demineralised water in a glass flask by stirring at room temperature for 30 minutes. While stirring, argon is bobbled through the monomer mixture in order to remove oxygen (O2). The coated radiofrequency chips are added to the monomer mixture, which is then transferred to a syringe pump. The oil is pumped from the glass flask with the use of a stepper motor to a first inlet of a first T-piece through a 3.0 mm inner diameter tube at a flow rate of 1000 mL per hour. The monomer mixture is pumped to the second inlet of the first T-piece at a flow rate of 10 mL per hour through a 0.3 mm inner diameter tube inserted in the exit tube from the T-piece such that the monomer mixture is Infused into the oil stream 5 mm downstream from the T-piece at which point ca. 1.0 mm diameter monomer droplets are generated inside the exit tube from the first T-piece. From the first T-piece the monomer-phase/oil-phase mixture is led to a first inlet of a second T-piece. A (10:1) mixture of oil and co-initiator (tetra-methyl-ethyl-diamine) is fed to the second inlet of the second T-piece. From the second T-piece the mixture is led through a 0.5 m long 3 mm inner diameter tube through an oil bath at 50° C. where the monomer droplets polymerise. The resulting beads and the oil are gathered in a glass flask at 50° C. and stirred. The beads are washed as described in example 5.
Example 11. Batch-wise preparation of radiofrequency tagged polymer beads 1000 radiofrequency chips (ca. 0.5 mm x 0.5 mm x 0.2 mm, produced by Hitachi, Japan) with external antennas operating at 2.45 GHz were purchased from Fine Technologies a.s., Oslo, Norway. 140 of the chips were removed from their external antennas. Thus the resulting chips had no antennas and therefore their radiofrequency codes could not be read,
However, the chips are useful for demonstrating the preparation of spherical radiofrequency tagged beads. Readable radiofrequency tagged beads can be prepared by the method described in the present example by using radiofrequency microchips with internal antenna, such as the mu-chips produced by Hitachi.
140 radiofrequency chips were removed from their antennas. 5 mL concentrated sulphuric acid was added to 5 mL 35% hydrogen peroxide, and the radiofrequency chips were added to the resulting hot solution. The chips were left in the solution for 30 minutes. The chips were then removed from the solution and transferred to warm (50° C.) demineralised water. After 2 hours in the warm water the chips were removed from the water.
5000 mL mineral oil was transferred to a cylindrical 8 L stainless steel reactor equipped with a stirring rod connected to a motor. The oil was heated to 70° C. 1.25 g of surfactant was added to the oil phase. The oil-surfactant mixture was purged with argon in order to remove oxygen (O2), 125 g of a macro-monomer mixture comprising ca. 50% polyethyleneglycol-di-acrylamide (molar mass ca. 1900 g/mole) and ca. 50% polyethyleneglycol-acrylamide (molar mass ca. 1900 g/mole) was dissolved in 375 mL demineralised water in a glass flask. 6 g acrylamide was added to the flask. 1.4 g ammonium persulfate was added to the flask. The radiofrequency chips were added to the flask. The aqueous monomer-radiofrequency chip mixture was stirred while argon was bobbled through it. The stirring of the oil in the reactor was started by switching on the motor connected to the stirring rod. The monomer-radiofrequency chip mixture was added to the steel reactor. After one minute 6.25 mL tetra-methyl-ethyl-diamine was added to the reactor. The mixture was stirred in the reactor at 70° C. for 45 minutes.
The resulting polymer beads were separated from the oil by filtering and then washed sequentially with dichloromethane, tetrahydrofuran, methanol, and demineralised water, The washed beads were sieved. Direct visual inspection of the size fraction larger than 1 mm diameter revealed that the radiofrequency chips had become fully encapsulated in polymer.
The density of primary amine (—NH2) groups in the polymer was measured by standard Fmoc method. The density was found to be approx. 0.2 mmol primary amine per gram of dry polymer.
20 radiofrequency chips of the same type as used in example 11 are treated with aqueous sodium hydroxide, washed with demineralized water, and submerged in (3-acryloxypropyl)-methyldimethoxysilane for 1 hour. The encapsulation of the radiofrequency chips in polymer is performed as described in example 11.
Example 13. Up-scaled preparation of radiofrequency tagged polymer beads 10,000 (ten thousand) radiofrequency chips with internal antenna (mu-chip, 0.4 mm×0.4 mm, by Hitachi) are surface treated and embedded in polymer as described in example 11.
The concentration of primary amine in the resulting polymer gel is ca. 0.2 mmol per gram of dry polymer, which corresponds to ca. 0.02 mmol per mL of water-swollen resin. A spherical bead of 2 mm diameter when swollen in water has a volume of ca. 0.004 mL and a total amount of primary amine of ca. 80 pmole.
The primary amine groups serve as starting points for solid phase combinatorial synthesis.
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2006 00059 | Jan 2006 | DK | national |
| Number | Date | Country | |
|---|---|---|---|
| 60757858 | Jan 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12160306 | Sep 2008 | US |
| Child | 13353141 | US |
