Patent Application
20050014146
The present invention relates to a method of molecular biological analysis utilizing dielectrophoretic forces to manipulate biological components advantageously and with high processivity. In particular, the method disclosed can be used to check the binding force between proteins and/or verify the presence and quantity of proteins in a sample, to assemble arrays of test points, to check the concentration of the proteins being tested, and, optionally, to observe the results with the aid of sensors integrated into the test device. The invention relates similarly to a device for implementation of the method thus outlined, equipped with the aforementioned integrated sensors.
A great many immunological methods have been developed in recent years allowing the determination of antigens and antibodies, both for purely scientific and for diagnostic purposes.
Immunoassays
Immunological tests, or immunoassays, utilize a number of notably powerful methods for identifying and measuring antigens and antibodies. Specific antibodies are available for an increasing number of antigens, soluble, immobilized (on plates, resins or membranes), conjugated and otherwise. Moreover, with the range of systems for analyzing antigen-antibody complexes becoming steadily wider, and their sensitivity continuing to be improved, the potential and the range of applications for immunological reactions and techniques have been extended conspicuously. In the case of soluble antigens and antibodies, assays are based on the labelling of one of the reagents, on the formation and precipitation of immunocomplexes, or on the measurement of an effector function expressed by the antibody.
For some time, the most sensitive system available was radioimmunoassay (RIA), developed by Yarlow and Benson in 1960. This method betrays numerous drawbacks at all events, including the need for special equipment, also for special precautions against radiation (and for specially trained staff), and the limited average life of the radioactive isotopes used for labelling purposes. Such constraints soon led to the notion of replacing isotopes with enzymes as the labelling medium. The first studies on Enzyme Immunoassay (EIA) were conducted by Schuurs et al. and disclosed in a series of patents: U.S. Pat. Nos. 3,654,090; 3,791,932 and successive references. EIA methods include ELISA (Enzyme-Linked ImmunoadSorbent Assay) and its numerous variations, which currently are the methods of choice in the art fields of research and diagnostics. EIA-ELISA procedures are categorized as competitive and non-competitive, which in turn can be homogeneous or heterogeneous. Whilst homogeneous assays require no physical separation, heterogeneous assays require separation of the free antigen fraction from the fraction bound to the antibody, obtained by means of a solid phase system consisting generally in polystyrene, cellulose or nylon substrates to which the antibodies are bound. The substrates are usually 96- and 384-well microtiter plates or microstrips having 8, 12 and 16 wells, though they can also consist in single elements known as microbeads, on which the antigens or antibodies are immobilized. Competitive enzyme immunoassays are those where the antibody is present in a limited concentration. In non-competitive or immunometric assays, on the other hand, a notable excess of the antibody is used, conjugated with the enzyme, so as to maximize the antigen signal. Among non-competitive enzymatic immunoassays, the system most widely adopted involves capturing antigens from the sample on the walls of microsites coated with antibodies, generally monoclonal (mAb). The captured antigen is marked by coating it with a second layer of specific antibodies (secondary antibodies) with or without further amplification steps. The secondary antibody is often conjugated with an enzyme, the conversion of the enzyme demonstrating the presence of a given antigen: this is known as a sandwich ELISA assay.
With a wide range of substrates available for marker enzymes, it is possible to choose between different detection methods. The substrates are reagents that allow of displaying, qualifying and/or quantifying an analyte of interest in an enzyme immunoassay. Substrates can be chromogenic, chemiluminescent or fluorescent. Chromogenic substrates produce a coloured compound that can be identified visually and quantified with a spectrophotometer. Chemiluminescent substrates produce light that can be measured with a luminometer or recorded permanently on X-ray film. Fluorescent substrates on the other hand emit fluorescence that is measured with a fluorometer. Chromogenic and chemiluminescent substrates are excellent media for the detection of conjugates labelled with enzymes bound indirectly to a solid support. The enzymes commonly used for the purpose are peroxidase, generally Horse Radish Peroxidase (HRP), which catalyzes the fission of H2O21 Alkaline Phosphatase (AP), which removes the phosphate from phosphorylate molecules, and β-galactosidase (β-Gal), which hydrolyzes lactose. The conversion of numerous substrate molecules by a single enzyme molecule produces a notable amplification of the signal, though if a luminogenic or fluorogenic substrate is used, the signal/mass is still greater, comparable to that obtained with Radioimmunoassays.
EIA methods are powerful, but affected by the serious limitation of low productivity (given the difficulty of conducting significant numbers of analyses in parallel), due mainly to the scant possibilities for integration afforded by the various items of equipment needed to carry out the procedure. This makes it all but impossible to process thousands of samples simultaneously or at least in a short time, whereas speed is becoming more and more a fundamental aspect of modern research and diagnostics. In addition, EIA can involve a relatively heavy consumption of costly reagents.
Labelled Microbeads
Not least in order to overcome the aforementioned drawbacks, the use of microbeads labelled selectively employing various fluorescence methods is gaining more and more importance in the art field of biotechnologies. Especially pertinent in this field are the following patents:
Molecular Sensors Based on Surface Plasmon Resonance
U.S. Pat. No. 5,641,640 in the name of BIAcore AB, discloses a system for the analysis of biological samples using surface plasmon resonance. Molecules of a sample held in suspension are directed into a chamber, of which the surface carries immobilized molecules potentially capable of binding with those of the sample. The binding of the molecules is sensed by indirect measurement of the variation in the refraction index caused by the binding of the molecules with the surface, observing the reflection from the surface of a suitable light source.
Dielectrophoresis
Dielectrophoresis relates to the physical phenomenon whereby dielectric particles subject to spatially non-uniform d.c. and/or a.c. electric fields undergo a net force directed toward those regions of space characterized by increasing (pDEP) or decreasing (nDEP) field strength. If the strength of the resulting forces is comparable to the force of gravity, it is possible in essence to create an equilibrium of forces enabling the levitation of small particles. The strength, direction and orientation of the dielectrophoretic force are heavily dependent on the dielectric and conductive properties of the body and of the medium in which it is immersed, and these properties in turn vary with frequency.
Studies analyzing the effects of dielectrophoretic forces on particles (the term “particles” is used hereinafter to indicate dielectrophoretically manipulated bodies or elements) consisting in biological entities (the term “biological entities” is used hereinafter to indicate cells and microorganisms, or parts thereof, namely DNA, proteins, etc.) or artificial objects consisting of inorganic matter, have suggested for some time the notion of exploiting these forces as a means of selecting a particular body from a sample containing a plurality of microorganisms, characterizing the physical properties of microorganisms and in general allowing their manipulation. Accordingly, it has been found advantageous to utilize systems comparable in size to those of the microorganisms being manipulated, and thus reduce the magnitude of the voltages used to create the field distributions needed to reveal the aforementioned effects.
Particles exposed to the phenomenon of dielectrophoresis are subject to forces dependent on the volume of the particle; this being the case, it has been assumed for some time that there must be a lower limit for particle size, beneath which dielectrophoretic force would be defeated by Brownian movement. It was considered that there would be a need for electric fields of magnitude such that local warming of the fluid would increase local flow and effectively prevent dielectrophoretic manipulation. Pohl (1978) speculated that the electric fields needed to trap particles smaller than 500 nm subject to Brownian movement would be too strong. The first group to overcome this obstacle was that of Washizu (Washizu et al., Trans. Ind. Appl. 30:835-843, 1994), who used positive dielectrophoresis to precipitate small proteins down to 25 kDa. This lowering of the threshold was favoured by improvements in electrode manufacturing technologies, notably the use of electron beams in manufacture. Thereafter, Fuhr et al. (Fuhr, 1995, Proc. St Andrews Meeting of Society for Experimental Biology p.77; Mueller et al., 1996, J. Phys. D: Appl. Phys. vol.29:340-349) and Green et al. (Green et al., 1995, Proc. St Andrews Meeting of Society for Experimental Biology p.77; Green et al., 1997, J. Biochem. Biophys. Methods vol.35:89-102) demonstrated that viruses of 100 nm diameter could be manipulated employing negative dielectrophoresis. It was also shown that latex microbeads of 14 nm diameter could be trapped both with positive and with negative dielectrophoresis (Mueller et al., 1996, J. Phys. D: Appl. Phys. vol.29:340-349). Subsequent studies showed that 68 kDa molecules of the protein avidin can be concentrated from solution using both positive and negative dielectrophoresis (Bakewell et al., 1998, Proc. 20th Ann. Int. Conf. IEEE Eng. Med. Biol. Soc. 20, 1079-1082).
Patent application PCT/WO 00/47322 discloses an apparatus and a method for manipulating particles utilizing closed dielectrophoretic potential cages, generated by singly and selectively addressable and mutually energizable adjacent electrodes making up an array.
Patent application PCT/WO 00/69565, filed by the same applicant, discloses a more efficient apparatus than that mentioned above and describes various methods of manipulating particles utilizing closed dielectrophoretic potential cages. The device described in this second PCT application is illustrated in
The electrodes of the array can be of any given shape; in the example of
The region beneath the electrodes (C in
In a preferred embodiment, the second main module appears substantially as a single large electrode M2, covering the device in its entirety. Finally, the device may also include an upper support structure (O2 in
The most suitable material for the upper electrode M2 will be a transparent conductive material. Besides allowing the inclusion of sensing circuits as outlined previously, this will also allow the use of traditional optical inspection means (microscope and TV camera) located above the device.
Among the singular aspects of the invention disclosed in patent application PCT PCT/WO 00/69565, parts of which are incorporated into the present specification where necessary for reference purposes, is that the one substrate can accommodate both the elements capable of manipulating the particles (biological entities), and the sensing devices.
The object of the present invention is to overcome the drawbacks inherent in the prior art methods outlined above for conducting biomolecular tests on biological entities (cells, microorganisms or parts thereof, in particular oligonucleotides, proteins or parts thereof) in such a way that these tests can be carried out swiftly, efficiently and economically, with precision and high processivity, using smaller quantities of reagents and especially of costly reagents, namely monospecific antibodies, labelled antibodies and substrates.
Here and in the following description, the term “protein” is used to indicate a molecular chain of amino acids bound by peptide bonds; the term does not refer to a specific length, and accordingly, the commonly used terms “polypeptide”, “peptide” and “oligopeptide” are also included in the definition. Also included are post-translational modifications of protein such as glycosilations, acetylations, phosphorylations and the like. Moreover, the term protein likewise includes protein fragments, analogues, mutated or variant proteins, fusion proteins, and so forth.
Just as the term antibody can be taken, where not explicitly stated, to mean antibodies obtained from polyclonal and/or monoclonal preparations, it can also be taken to mean chimeric antibodies, F(ab′)2 and F(ab) fragments, Fv molecules including single chain (sFv), dimeric and trimeric constructs of antibody fragments and any fragment obtained from these and similar molecules, where these happen to maintain the specific binding properties of the original antibody molecule.
In the light of the foregoing definitions, one object of the present invention in particular is to exploit the potential afforded by the device of patent application PCT/WO 00/69565 in providing a method of conducting integrated biomolecular analysis on a biological sample including unknown biological entities, for example specific proteins or antigens or specific antibodies, by means of known biological entities, typically antibodies, or natural or synthetic proteins, such as can be run with a high level of automation and in parallel, if necessary, on a high number of samples, or on a significant number of different biological entities in one sample.
The stated objects are realized in a method according to the present invention for conducting integrated biomolecular analyses on a biological sample including unknown biological entities, with the aid of known biological entities capable of binding to the unknown biological entities, comprising the steps of immobilizing first biological entities directly or indirectly on a support, bringing second biological entities into contact with the first and detecting any binding activity between at least a proportion of the first biological entities and at least a proportion of the second biological entities; the first or second biological entities being the unknown entities and the second or first biological entities being the known entities; characterized:
One of the singular features of the method according to the invention consists moreover in the facility of concentrating antigens and/or antibodies involved in the analysis by attracting them into the dielectrophoretic cages. Other characterizing features of the method disclosed include the facility of generating protein microarrays, by dielectrophoretic manipulation of the protein population of interest, which can then be assayed to reveal their affinity with other proteins (antigens or antibodies). Moreover, the specificity of the antigen-antibody bond can be tested electronically by trying to separate the bound proteins, seeking to draw one of them back into the dielectrophoretic cages by varying the particular force and/or frequency of the cage. The test can be monitored exploiting standard methods (fluorescence, luminescence or colour development) and employing optical sensors, which can be external (microscope and TV camera) or integrated into the device. Alternatively, it is possible to use a method exploiting capacitive sensors integrated into the device to observe the formation of antigen-antibody complexes.
A further object of the invention is to provide a device for conducting molecular biological analyses that will be notably compact, economical and reliable, while capable of fully automated operation and processing at high speed.
The stated object is realized according to the present invention in a device for molecular biological analyses performed with the aid of movable dielectrophoretic cages, comprising a surface afforded by an array of first electrodes selectively energizable and addressable at least in part and arranged on an insulating support; at least one second electrode positioned opposite and facing at least a part of the array of first electrodes; and a spacer serving to distance the first electrodes from the at least one second electrode in such a way that the second electrode, the spacer and the array of first electrodes combine to establish a test chamber encompassing a liquid or semi-liquid environment; characterized in that it further comprises integrated optical sensors located beneath or in close proximity to at least one of the first electrodes; and in that the first electrodes comprise means by which to allow the transmission of electromagnetic radiation through the selfsame first electrodes and toward the optical sensors, operating in conjunction with means likewise forming part of the device and positioned to coincide with the first electrodes, by which radiation of a first predetermined wavelength is prevented from reaching the integrated optical sensors.
The advantages of the present invention are many and various.
The proposed method guarantees high sensitivity thanks to the possibility of concentrating the protein populations present in samples by attracting them selectively into the dielectrophoretic cages. This naturally signifies a saving in expenditure on reagents, as well as the facility of testing samples to the limit of the detection potential afforded by standard methods.
Another singular advantage is the facility of verifying the specificity of the assay by way of an electronic antigen-antibody binding affinity check, which will eliminate false positives generated by possible cross-reactivity of the antibodies, a likelihood that cannot be excluded when handling thousands of antigens or antibodies together. This procedure also allows the stability of the antigen-antibody bond to be evaluated directly.
Complementing the high sensitivity obtainable with the method according to the present invention is an appreciable parallelism, given that the assay can be conducted on all the proteins in a single chamber rather than in a plurality of distinct, albeit very similar chambers. This, together with the high level of integration and feedback control achievable thanks to the automation allowed by the device and the method disclosed, means that any variability of response given by the assay due to system-related and/or accidental (operator) errors can be reduced to a minimum. Another advantage of the method is that of integrated sensing, which dispenses with the need for cumbersome instruments (fluorometers, luminometers, etc.), which very often are not even associated with the test device. In the case of direct capacitive sensing, the experimenter avoids the need for labelling of the antibodies employing generally complex and costly procedures, to facilitate their identification. Likewise in the case of capacitive (indirect) labelling by means of microbeads, the procedure is particularly simple and applicable even to antigenic proteins.
In the case of a directly assembled protein array, exceptionally high density is achievable given that thousands of different proteins can be patterned on the electrodes of the device, which are spaced at a particularly fine pitch.
Other features and advantages of the invention will emerge more clearly from the following description of certain preferred embodiments illustrated by way of example, and implying no limitation, with the aid of the accompanying drawings.
With reference to
The method according to the present invention is carried into effect, unless otherwise indicated, employing conventional chemical and biochemical procedures commonly used and widely documented in literature. The preferred procedure, though not exclusive and implying no limitation whatever, is that illustrated in
The procedure begins with construction of the protein array to be tested; the array in the example of
Alternatively, the protein array to be tested on the electrodes can be prepared using standard microarray technology, such as ink-jet.
At this point the sample containing the biological entities to be tested (mixture of antibodies) is introduced into the device (
The test can be monitored exploiting methods that use fluorescence, chemiluminescence, etc. In the example of the drawing, an antibody population is labelled with a fluorescent marker molecule (
An alternative option would be to use a method exploiting capacitive sensors integrated into the device (conventional in embodiment and therefore not illustrated), such as will indicate the capacitance associated with the electrode of each single protein site established previously and show the difference in capacitance when another protein binds to those already present at the site (
The variation in capacitance can be identified employing the methods and circuits disclosed in patent application PCT/WO 00/69565.
One variation on the method according to the present invention relates to a test procedure in which the sample containing the proteins to be identified is immobilized in spatially uniform manner on the surface of the device, above the electrodes, as indicated schematically in
The antibody-protein binding check is run simply by resetting the frequency to f1; if binding has occurred, the microbead will not be able to return inside the cage (
Clearly, the microsupport selected for immobilization of the biological entities to be manipulated and/or identified can be a medium other than a microbead; for example, the molecules of interest might be immobilized on the surfaces of cells or liposomes.
In accordance with a further variation on the method, moreover, the antigen-antibody binding force check can be run without using dielectrophoresis, but simply introducing a flow of buffer solution into the environment L, directed through the surrounding chamber; in this instance it will be hydrodynamic force that induces the bound biological entities to separate from the surface afforded by the electrodes LIJ.
To enable the detection of fluorescent marker molecules, whether associated directly with the biological entities or with microbeads (or with other microsupports as mentioned above), the device of
In accordance with the state of the art, the typical excitation wavelengths for these molecules range from 350 to 480 nm for Ar, Xe—F and Xe ion lasers. It is therefore important that the optical sensors incorporated into the substrate C should be selective, in particular, not liable to react to ultraviolet radiation, and especially sensitive to radiation in the visible spectrum. This performance potential can be delivered by employing suitable techniques for the embodiment of semiconductor type optical sensors, which also constitute subject matter of the present invention, as will now be explained.
In general, a photon related to the ligh flux LIG (
One method commonly utilized to quantify photogenerated charges, and thus measure the intensity of the photon stream, consists in establishing a reverse biased p-n junction (XJ or XJW) in the region through which the flux is directed. A device embodied in this fashion is known as a photodiode, denoted CPH in
Utilizing planar technology, the foregoing operations are implemented according to the present invention by placing a contact CON on the diffusion surface of the photodiode CPH, such as can be connected electrically by way of an electronic address switch SW to the input of an electronic charge amplifier CHA. The output OUT of the charge amplifier encodes the amount of charge and therefore the luminous intensity incident on the photodiode CPH. It is possible to demonstrate that the space-charge region is the main factor responsible for photogeneration current.
The response of the photodiode as a function of the wavelength of the incident radiation thus depends to a considerable extent on the depth DEP of the junction (
Current MOS planar technology affords different possibilities for the manufacture of photodiodes: in particular, the preferred solution consists in diffusion using shallow-junctions and well-junctions.
In conclusion, the use of a deep well junction is particularly suitable for the proposed application, in order to eliminate the influence of ultraviolet radiation while maintaining good sensitivity at visible wavelengths.
Another way of increasing the selectivity of the sensors or more simply ensuring a higher level of confidence when using surface junctions (such as those deriving from the most sophisticated technologies), obtainable following procedures already familiar in the art field of semiconductor device manufacture, is that of utilizing suitable colour filters GEL deposited on the surface of the substrate C. These filters can be overlaid on the chip by means of photolithography and consist in colour photoresists or gels characterized by deposition resolutions of a few tenths of one micrometre (μm). In the example of the present disclosure, any ultraviolet interference can be reduced by using filters tuned in the yellow or green colour range.
In one possible embodiment of optical sensing means according to the invention, the p-n junction XJ or XJW is located in the silicon region C beneath the electrode LIJ, the electrode being fashioned photolithographicaly from materials that are electrically conductive, but transparent, typically Indium Tin Oxide (ITO). This solution can be obtained by post-processing an integrated circuit produced using the standard silicon technology applied routinely in microelectronics manufacturing processes, whereby the final passivation layer is applied in such a way as to leave portions of the metallization raised and exposed. The metallization is then used to establish an electrical contact between the transparent electrode and the circuits beneath.
In other solutions, utilizing an electrode LIJ of conventional embodiment that may not be transparent to light radiation, the photodiode could be located in the substrate, occupying the gap between the single electrodes, and the signals selected in such a way as to position the potential cage exactly in the space above the gap. In a further possible solution, electrodes embodied in non-transparent material could be fashioned with a central window, as mentioned previously, through which light can be directed so as to fall on the substrate beneath incorporating a photodiode.
Lastly, another way of preventing radiation emitted at the first frequency (UV in the example illustrated) from falling on the photodiode, is to create a waveguide utilizing the oxide of the chip and the glass of the lid, which will allow the fluorophores in the sample to be excited by radiation at a first frequency, directed laterally into the chamber holding the sample. The waveguide created in this manner will prevent the excitation energy from penetrating the substrate, since the unwanted radiation is reflected from the surface of the array by reason of its minimal angle of incidence, whilst that emitted by the fluorophores at given points of the array, being omnidirectional, will penetrate the surface of the array.
| Number | Date | Country | Kind |
|---|---|---|---|
| TO01 A 000801 | Aug 2001 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IT02/00524 | 8/7/2002 | WO |
