Patent Application
20100060893
The present invention is directed toward an apparatus and method for concentrating and imaging particles associated with certain assays.
Particles and magnetic, paramagnetic or superparamagnetic particles in particular, may be used in diagnostic assays as solid phase capture or detection species. Microparticle-based assays can be divided into two main categories: homogeneous (separation-free) and heterogeneous assays.
In a homogeneous (separation-free) assay format, binding reactants are mixed and measured without any subsequent washing step prior to detection. The advantages of such a system are fast solution-phase kinetics, a simple assay format, simpler instrumentation as well as lower costs because of fewer assay steps, low volumes and low waste. Homogeneous immunoassays do not require physical separation of bound and free analyte and thus may be faster and easier to perform then heterogeneous immunoassays. Homogeneous immunoassay systems using small sample size, low reagent volume and short incubation times, provide fast turnaround time. Disadvantages of this type of assay can be limited dynamic range and sensitivity. Since there is no separation of free analyte before signal detection, sensitivity might be further compromised. Also, interferences could cause a high background signal by interaction between the sample and capture or detection reagents. Homogeneous assays are the preferred assay format in high throughput screening platforms such as AlphaScreen, SPA, fluorescent polarization and flow cytometry based assays, as well as in diagnostic assays such as particle agglutination assays with nephelometry or turbidimetry as the detection methods.
Various types of assay involve the association of optically labeled detection particles with magnetic capture particles. When magnetic capture particles are used in a homogeneous (separation-free) assay format, the behavior of the particles in a magnetic field can be utilized to concentrate or differentiate the magnetic particles and anything which might be bound to them from other components of the assay. In assay implementations where the capture particles are bound or otherwise associated with optically labeled detection particles, it is difficult using known technologies to concentrate the capture particles at the focal plane of an optical interrogation system. Further difficulty is introduced if it is desired that the magnetic capture particles be concentrated in a relatively compact and/or small pellet for interrogation.
The present invention is directed toward overcoming one or more of the problems discussed above.
One embodiment includes an assay apparatus having a sample vessel within which an assay may be performed. The apparatus further includes a holder having a receptacle, socket or other device configured to operatively receive the sample vessel in a precise and easily repeated location with respect to the holder. This embodiment further includes a magnet operatively associated with the holder such that a magnetic field generated by the magnet intersects a portion of the sample vessel defining a magnetic concentration region within the sample vessel. The magnet may be a permanent magnet or an electromagnet. This embodiment may be used with a separate detection or interrogation instrument, typically a spectrometer. The spectrometer may also include apparatus specifically configured to receive and hold the sample vessel such that the focus of the spectrometer is within the magnetic concentration region of the sample vessel.
The various embodiments of the assay apparatus described herein are suitable for use with any assay where optically labeled particles become associated with magnetic, paramagnetic or superparamagnetic capture particles. The term magnetic particles as used herein includes conventional magnetic particles, paramagnetic particles, superparamagnetic particles or any other type of particle affected by a magnetic field. In one embodiment which is described in detail herein, the spectrometer is a Raman spectrometer and the optically labeled detector particles are SERS nanotags.
An alternative embodiment includes a sample vessel, holder and magnet defining a magnetic concentration region as described above. This embodiment further includes a spectrometer operatively associated with the holder such that the focus of the spectrometer is within the magnetic concentration region when a sample vessel is placed into or on the holder. In this aspect of the present invention, an intermediate step of moving the sample vessel from the holder/concentrator to a separate spectrometer is eliminated. Two significant advantages are achieved with an embodiment with combined functions. The first advantage is improved accuracy in placing the magnetic particles (or defining the magnetic concentration region) within the sample vessel, such that measurement repeatability is improved, and the possibility for false negative results (missing the concentrated pellet of magnetic particles altogether) is avoided. The second advantage concerns eliminating the step of moving the sample vessel from the holder/concentrator to a separate spectrometer, thus sample reading is reduced to a single step operation. Furthermore, the associated risk of movement of the pellet within the sample vessel or disassociation of the pellet during transfer to the spectrometer, which may cause errors in measurement, is eliminated.
Any embodiment of the apparatus described herein may further include a device associated with the sample vessel to stimulate relatively compact concentration of the magnetic capture particles within the magnetic concentration region. Alternatively, certain methods described herein may be used to assist with compaction and concentration. Representative devices which can be used to stimulate relatively compact concentration include but are not limited to: a magnetic stimulator which operates by passing a moving magnetic field through a portion of the sample vessel; a mechanical or electromechanical stimulator or vibrator configured to shake, agitate or vibrate the sample vessel; an acoustic transducer which might be an ultrasonic transducer configured to transmit acoustic waves through the sample vessel. Suitable compaction methods include the use of the devices described immediately above plus manipulation of the vessel in order that the pellet is forced to re-form at a different location on the vessel wall. For example, manipulation may include removal of the tube from the magnetic field and its replacement at an alternative orientation, or removal and reapplication of the magnet or magnets at a different location on the tube. In addition, the compaction enhancement methods can be applied with or through a separate localizing apparatus, or an apparatus that includes a spectrometer or other interrogation device.
Further embodiments include methods of performing an assay. Methods within the scope of the present invention include associating magnetic capture particles with optically active detection particles in a sample vessel, magnetically concentrating the magnetic capture particles in a concentration region of the sample vessel, and obtaining a spectrum from the magnetic concentration region utilizing apparatus as described above. The methods may further include stimulating compact concentration by mechanically agitating, vibrating, projecting acoustic waves, passing a moving magnetic field through a portion of the sample vessel, manipulating the sample vessel, rearranging the magnetic field or other means.
In each embodiment disclosed it is advantageous to minimize the amount of assay fluid which is outside of the concentration region, but along the optical path between the spectrometer and the concentration region. Apparatus and techniques disclosed to achieve this goal include but are not limited to positioning the various components so that the magnetic concentration region is caused to form where the optical path initially intersects the sample holder.
Various assays featuring the use of magnetic capture particles and optically labeled detection particles which assays may be implemented with the apparatus and methods described herein are fully described in co-pending application no. PCT/U.S.07/61878 entitled “SERS NANOTAG ASSAYS”, which application is incorporated herein in its entirety.
As used throughout this application, the term “magnetic particles” shall be defined as conventional magnetic particles, paramagnetic particles, superparamagnetic particles or any other particle which is affected by a magnetic field. Certain advantages may be realized by having an apparatus for the capture and concentration of magnetic particles used with an assay where the capture and concentration apparatus is separate from the associated optical or other detection/interrogation instrumentation. For example, it may desirable to minimize the time occupied by individual samples in the optical interrogation unit. Alternatively, advantages may be realized by having magnetic capture and concentration apparatus integrated with a spectrometer or other optical interrogation device. For example a fully integrated capture, concentration and imaging system minimizes the possibility of the misplacement or de-compaction of a pellet of concentrated magnetic particles since the sample vessel does not have to be moved between the concentration and optical interrogation steps. Accordingly, both separate and integrated apparatus embodiments are described in detail below. The types of apparatus described herein are not mutually exclusive embodiments. Features of the various types of apparatus described may be combined in hybrid apparatus to meet the specific needs of a user.
Also shown in
Although a spherical magnet is shown in
Additionally, sample vessels having any number of customized shapes may be used to effectively implement the apparatus. The customized shape might include a protrusion or indentation or other structure or form at or near the magnetic concentration region 20 described above. The customized shape may affect the nature of the pellet formed in the concentration step. The development of a concentrated, well-formed, and consistent magnetic pellet from magnetic capture particles and associated optical labels is a function of the magnetic field architecture as well as the sample tube morphology at the magnetic concentration region 20. Thus, the size and shape of the concentrated pellet can be altered by altering the shape of the surface the magnetic capture particles are collected upon or within. Therefore, customized tube architecture with a dimple (or cusp) or other shaped structure or form at or near the magnetic concentration region could aid in the formation of a magnetic pellet having reproducible size, shape and consistency. For example, a concave cusp may be utilized to form a spherical pellet at the bottom or side of a customized collection vessel.
Other shapes or forms associated with a vessel may be devised to facilitate the formation of a concentrated pellet of select shape and size. Thus, the sample vessel 14 has 4 primary functions:
The material of the sample vessel 14 must be compatible with the reagents and analyte substances intended for use in the assay. Certain chemical or biological coatings may be applied to the vessel to prevent non specific binding of magnetic particles, optical tag or analyte to the vessel walls. The sample vessel 14 should be constructed of a non-magnetic material, such that a magnetic field applied from an external source passes through the sample vessel 14 unaffected. Furthermore, it should be designed such that consideration is given to the maximum distance that would be traveled by reagents to the localisation spot. This governs the speed and efficacy of magnetic capture, since the attractive force decreases rapidly as the distance from the magnet system is increased. Additionally, the shape of the sample vessel 14 can be useful in concentration of the particles and pellet formation, through the overall shape of the vessel or through a special structure such as a dimple or cusp as described above. The sample vessel 14 also provides a means for accurate placement in holders for magnetic concentration, or spectroscopic measurement.
The form of the sample vessel 14 may include external geometric features, markers or indicia 22, to enhance the repeatability of positioning, and prevent incorrect vessel insertion within the holder apparatus. Positioning features could include (but are not limited to) the inclusion of flat sections, keyways, fiducial points etc. These would match similar features in a specifically designed vessel receptacle associated with magnetic localisation, spectroscopic measurement or combination of these functions, all as described herein.
In embodiments where assay interrogation is performed optically, typically a select laser output is applied to the concentrated pellet. Thus the sample vessel 14 should be appreciably transparent in the desired laser interrogation wavelength region. Moreover, it is desirable to ensure that the localizing region has optical properties compatible with the lens system of the spectrometer or other detection device. Flat optical surfaces that allow the entry and exit of excitation and emitted light with minimal refraction, reflection or scattering may be desired. Alternatively, surfaces with curvature in one or more dimensions intended to function as lenses may be beneficial. Furthermore, the vessel design should allow the simultaneous presence of a magnetic localizing system and operation of the spectrometer in the case where both functions are included in one instrument. The magnetic concentration region 20 can be at any position on the sample vessel 14 conducive to the ability to concentrate and localise the assay reagents and make optical measurements. This includes (but is not limited to) the bottom, sidewall or top cap of the sample vessel 14.
The various embodiments described herein are particularly advantageous for the concentration of magnetic capture particles associated with SERS nanotags or similar SERS taggants as the optically labeled detection particles. SERS nanotags have a SERS active core associated with a Raman reporter molecule which may be interrogated through Raman spectroscopy. Accordingly, it is useful in any implementation of the present invention which features Raman spectroscopy that the material of the sample vessel 14 be transparent to Raman light scatter. For example, the apparatus schematically illustrated in
In the arrangement shown in
As illustrated in
An alternative fully integrated magnetic concentration and imaging apparatus 38 is shown in the schematic diagram of
As is best shown in
Apparatus and methods where the amount of free assay fluid along the optical path is minimized are distinctly different from known devices where the spectrometer is positioned opposite the vessel from the magnetic concentration region. Optical interrogation through the assay medium as is typical with known devices can cause substantial read noise. In particular, read noise from unbound detection tags can be problematic in a case of a homogeneous (no wash) assay. Problems associated with unbound assays in the optical path can be eliminated or minimized by utilizing the novel configuration illustrated in
As shown in
Graph 46 of
Pellet formation can be achieved with a static positioning between the sample vessel 14 and magnet 18. However, the magnetic particles may be concentrated to a smaller volume if the magnet 18 is moved relative to the surface of the sample vessel—a displacement of less than 1 mm is sufficient to achieve measurable concentration improvement. Movement of the magnet 18, however, may be impractical, due to the requirement that the position of the magnetic concentration zone 20 be in coincidence with the laser illumination from the spectrometer 40. It is possible to stimulate tight pellet formation however, by use of techniques or methods including but not limited to mechanical, acoustic or magnetic stimulation of the sample vessel 14. Vibrational energy is sufficient to dislodge particles that have become stuck to the vessel wall before reaching the final position of lowest potential energy nearest the magnet 18 or magnetic pole piece 44. Practical methods by which this may be achieved include but are not limited to the following: Momentary vibration of the holder 12; momentary vibration of the sample vessel 14 within the sample holder 12; momentary exposure of the sample vessel 14 and contents to a high pressure acoustic wave; or the application of a moving or oscillating magnetic field, such as from an electromagnet. These methods introduce enough perturbation to facilitate the formation of a lowest energy state dense pellet. In addition, there is the further option of causing the pellet to reform by manipulation of the sample vessel 14 in order that the pellet is forced to re-form at a different location on the vessel wall 24, e.g. by removal of the tube from the magnetic field and its replacement at an alternative orientation, or by removal and reapplication of the magnet 18 or magnets at a different location on the sample vessel 14.
In certain embodiments of an integrated capture/interrogation unit 38 such as shown in
Although the embodiments of
With a non-integrated magnetic capture and concentration apparatus 10 such as shown in
A ½″ spherical NdFeB (rare-earth) magnet has been successfully used experimentally to create a pellet of magnetic beads and associated SERS tags in a single pull-down step. The sample vessel 14 used was a 200 uL polypropylene microcentrifuge tube with 100 uL or less of reagent. As shown on graph 46 of
In an alternative assay system embodiment, a whole pellet may be imaged using a tunable filter (LCTF, AOTF, etc.) to create a hyper-spectral image stack. If consistent pellet formation proves to be difficult, imaging the whole pellet combined with subsequent image analysis could provide a metric for a “total optical label” that is consistent without respect to the distribution or shape of the pellet.
Similarly, a diffuse pellet, for example a linear pellet formed by a linear magnet, could be adequate for a reproducible, homogeneous, magnetic pull-down assay if spatial information is captured by the means of a scanning or wide-field imaging system. In this embodiment, the pellet may be imaged and a software analysis step employed to determine a metric for the total signal (for example the SERS signal) emitted by each pellet. This implementation shares features with DNA microarray readers commonly available, except for the need for a relatively large number of spectral channels (i.e. hyperspectral imaging). Nonetheless, for a high-throughput system utilizing a microplate configuration with multiple wells, this could offer benefits. Optical resolution, on the order of 50 um, could be adequate to allow an image processing algorithm to account for pellet size and shape differences to obtain a consistent total signal metric across replicate pellets. Prior research shows that a minimum number of spectral channels (˜20-30 spectral channels), could be adequate to differentiate and quantitate SERS tags. In a wide-field embodiment this may provide for the use of liquid-crystal tunable filters (LCTF), or acousto-optical tunable filters (AOTF) to achieve spectral separation. With respect to a laser-point scanning system, a low spectral resolution dispersive spectrometer system could be adequate.
In a high-throughput detection system it may be preferable to separate the magnetic concentration step from the optical read step. Magnetic concentration freezes the chemical reaction and so all samples in a batch, for example all of the samples on a microwell plate, would require that their pellets be formed at the same time. Large process volumes may result in variation in the exact location of each pellet. One possibility to address this issue for high-throughput geometry, therefore, is to use an initial imaging step, not to gather spectral information, but merely to locate the pellets. An image processing step could extract the positions of all the pellets and use this information to position a conventional point spectrometer system under each pellet sequentially.
High throughput methods could include automated machinery to perform the following functions:
High throughput apparatus could be fabricated in which the sample vessels are discrete tubes. The tubes could be transported within the apparatus to various function “stations” by a conveyor track, belt or robotic system. The precise location and timing of each sample tube could be tracked by a microprocessor system that controls the apparatus function, enabling precise control of reaction timings, temperatures, mixing and measurement etc. Such an apparatus for the invention might include provision for process steps including but not limited to
The sequence of steps could include the methods of localisation separate from, or coincident with making optical measurements, in the same manner as described already, thus the high throughput apparatus would incorporate receptacles for the vessels that have magnet assemblies for localisation, and/or spectrometers operatively associated with magnet assemblies. Other variations upon the basic high throughput apparatus described herein are within the scope of this disclosure.
Another embodiment of the present invention utilizes a small bead composed of a material of high magnetic permeability (e.g. nickel, cobalt, etc) placed in the reaction vessel. The presence of the high-permeability bead will focus the magnetic field lines thus creating a high magnetic field gradient to attract the magnetic particles. The magnetic particle/assay tag complex could associate around the bead and the whole large assembly, consisting of a large “bead” with the surrounding small magnetic particles and assay tags would be imaged. This technique could assist in achieving a reproducible spherical pellet for optimal interrogation.
Another embodiment features automated positioning of the sample vessel 14 at the optical detector. This is a variation of a high-throughput method where readout is possibly performed serially but magnetic concentration can be accomplished in parallel. A set of sample vessels containing all pre-mixed reagents, perhaps introduced to the vessels through the use of an automated fluid handler system, would have a magnetic assembly to concentrate the magnetic particles with corresponding reagent. This would, in effect, stop the reaction, but would also be a coarse pull-down forming a large diversity of pellets. Each vessel could then be positioned in a holder 38 such as shown in
In certain embodiments it may be desirable in either a normal or high throughput system to eliminate the need for pellet re-forming or conditioning. One suitable apparatus for use without pellet conditioning is schematically illustrated in
The use of a capillary tube 52 as an alternative format sample vessel provides several distinct advantages including but are not limited to:
The capillary tube-based lateral flow embodiment described immediately above is suitable for use as an integrated assay system which could if desired be developed in a very compact form factor for highly accurate and repeatable field assay usage. An integrated system 60 consistent with this embodiment is schematically illustrated in
The integrated system 60 of
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/74161 | 7/24/2007 | WO | 00 | 11/17/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60832921 | Jul 2006 | US | |
| 60910246 | Apr 2007 | US | |
| 60910256 | Apr 2007 | US |
