Patent Application
20130337569
The present invention is directed toward a method and system for the use of Surface Enhanced Raman Scattering nanotags (SERS nanotags) to create a variety of assay platforms.
Particles, and magnetic particles in particular, are extensively 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.
Heterogeneous immunoassays requiring the separation of free analyte and of unbound detector and in certain instances may be more versatile than homogeneous assays. The wash or physical separation steps eliminate most interfering substances and in general do not interfere with the detection/quantification step. Stepwise heterogeneous assays are possible which allow for larger sample size, which in turn improves sensitivity and yields wider dynamic range than the standard assay curves. The disadvantages of heterogeneous immunoassays are that they are much more labor-intensive, time-consuming and typically require dedicated analyzers. In addition, automated heterogeneous systems require more complicated designs or multiple instruments to accommodate wash and separation steps. Many clinical analyzers use magnetic microparticles for heterogeneous diagnostic assays to selectively bind and then separate the analyte of interest from its surrounding matrix using a magnetic field (The Immunoassay Handbook (2001) Nature Publ, London). Analyzers based on this format include the ACS 180 and Immuno I from Bayer Diagnostics, the Access from Beckman Coulter and the Elecsys from Roche Diagnostics.
Traditionally, only one biomarker has been used for diagnosis of a disease with homogeneous assays such as described above: for example Prostate Specific Antigen (PSA) is the marker of choice for prostate cancer even though it may have poor specificity (Lu-Yao et al. (2003) J Natl Cancer Inst. 95:1792-1797). With the advent of proteomics, there is a belief that diagnostic assays involving several biomarkers will increase the accuracy, specificity and aid in discerning between different diseases that exhibit similar symptoms. Screening for several markers, as is already done in the genomics field, provides better diagnostic or prognostic information than screening for one biomarker alone. See for example, Genomic Health's Oncotype Dx Breast Cancer Assay (2005) (www.genomichealth.com).
Assays designed to shorten the time from sampling to diagnosis are equally important in emergency room or point-of-care settings. For example, Biosite Inc. offers a lateral flow immunoassay for the rapid diagnosis and assessment of the severity of heart failure and risk stratification in acute coronary syndromes (Biosite Website (2005) http://www.biosite.com/products/cardiac.aspx.) Simpler, more accurate, and faster assay technologies are therefore needed to clinically implement the many biomarkers newly identified through proteomics research. The ability to quantify three or more proteins simultaneously in a fast, no-wash, no-sample preparation bioassay would dramatically improve the state of the art in disease diagnosis and prognosis, as well as in disease progression monitoring.
The present invention is directed toward overcoming one or more of the problems discussed above.
The present invention provides several methods and systems for the use of Surface Enhanced Raman Scattering nanotags (SERS nanotags) to create homogeneous (no-wash), heterogeneous or sequence detection assay platforms. In certain embodiments the SERS nanotags are used in combination with magnetic particles. Multiplexed assay platforms are also disclosed. In certain embodiments, the assay is useful for clinical proteomics. Assay platforms suitable for use within a biological matrix, for example within whole blood or serum are also disclosed. The assay formats described herein may be used to detect any analyte of interest including but not limited to the detection of cells, viruses, bacteria, proteins, DNA, RNA, or small molecules in any type of biological (animal or plant kingdom) or environmental samples including but not limited to whole blood or serum, occult samples, urine, feces, air, drinking water, phage, any organism, multicellular clumps of cells, for example, cancer tissue homogenate.
The Detailed Description of the Invention below includes the following subsections:
The use of magnetic capture particles in certain of the assays discussed below will enable easy integration with existing clinical analyzers. The use of SERS nanotags (as will be elaborated on below) will enable the use of a no-wash, multiplexed, assay format. It is important to note that there is an industry wide emerging trend toward miniaturization, typically by use of microfluidic and nanofluidic technologies, which will impact the diagnostics field by allowing the reduction of reagent and sample volume as well as reducing the reaction time involved in each experiment. Miniaturization may thus potentially lower the total cost-per-experiment However, to achieve the benefits of miniaturization, new sets of challenges such as the need for precise and accurate liquid handling procedures must be overcome. The assay method and platform discussed herein in many instances requires no liquid handling, thus circumventing the micro and nano-fluidics issues. The assay methods and platforms of the present invention will handle and probe any volume size equally well.
A homogeneous multiplexed assay platform is typically designed around a two-particle system. A capture particle is conjugated with an antibody to capture the antigen of interest from the biological sample. The detection particle may be a SERS-nanotag particle as described below also conjugated with a detection antibody with binding affinity for the antigen of interest. The SERS nanotag includes a Raman reporter molecule as described below. In the presence of the antigen of interest both the capture particle and the detection particle are bound to form a SERS active, 2-particle, immunocomplex. SERS nanotags offer at least three intrinsic advantages as detection tags. (1) They can be excited in the near-IR, and thus are compatible with whole blood measurement. (2) SERS nanotags resist photobleaching which allows for higher laser powers and longer data acquisition times, resulting in more sensitive measurements. (3) A large number of distinct tags exist, enabling highly multiplexed assays.
SERS nanotags are novel, nanoparticulate optical detection tags based on surface enhanced Raman scattering (SERS) (Mulvaney et al. (2003) Langmuir 19:4784-4790; Natan, U.S. Pat. No. 6,514,767). Raman scattering (Long (2002) The Raman Effect; A Unified Treatment of the Theory of Raman Scattering by Molecules. John Wiley & Sons Ltd, Chichester; Modern Techniques in Raman Spectroscopy (1996) John Wiley & Sons Ltd, Chichester; Analytical Applications of Raman Spectroscopy (1999) Blackwell Science Ltd, Malden, Mass.) SERS is a laser-based optical spectroscopy that, for molecules, generates a fingerprint-like vibrational spectrum with features that are much narrower than typical fluorescence. Raman scattering can be excited using monochromatic far-red or near-IR light, photon energies which are too low to excite the inherent background fluorescence in biological samples. Since Raman spectra typically cover vibrational energies from 300-3500 cm−1, it could be possible to measure a dozen (or more) tags simultaneously, all with a single light source. However, normal Raman spectra are very weak, limiting utility for bioanalytical chemistry. In SERS, molecules in very close proximity to nanoscale roughness features on noble metal surfaces (gold, silver copper) give rise to million- to trillion-fold increases [known as enhancement factor (EF)] in scattering efficiency (Moskovits (1985) Rev. Mod. Phys. 57:783-826; Otto et al. (1992) J. Phys. Cond. Mat. 4:1143-1212; Campion and Kambhampati (1998) Chem. Soc. Rev. 27:241-249; Tian et al. (2002) J. Phys. Chem. B 106:9463-9483; CAS Online Search, April 2004), More importantly, SERS can also be used to detect molecules adsorbed to individual metal nanoparticles (Emory et al. (1998) J. Am. Chem. Soc. 120:8009-8010; Moyer et al. (2000) J. Am. Chem. Soc. 122:5409-5410), and has been used to demonstrate detection of single molecules (Nie and Emory (1997) Science 275:1102-1106; Kneipp et al. (1997) Phys. Rev. Lett. 78:1667-1670; Michaels et al. (1999) J. Am. Chem. Soc. 121:9932-9939; Xu et al. (1999) Phys. Rev. Lett. 83:4357-4360; Goulet et al. (2003) Anal. Chem. 75:1918-1923).
A typical SERS nanotag 10 is shown in
The nanoparticle core 12 may be of any material known in the art to be Raman-enhancing. The nanoparticle cores 12 may be isotropic or anisotropic. Anisotropic nanoparticles may have a length and a width. In some embodiments, the length of an anisotropic nanoparticle is the dimension parallel to the aperture in which the nanoparticle was produced. In the case of anisotropic nanoparticles, in some embodiments, the nanoparticle has a diameter (width) of 350 nm or less. In other embodiments, the nanoparticle has a diameter of 250 nm or less and in some embodiments, a diameter of 100 nm or less. In some embodiments, the width is between 15 nm to 300 nm. In some embodiments, the nanoparticle has a length of about 10-350 nm.
Nanoparticles suitable to be the core of a SERS nanotag include colloidal metal, hollow or filled nanobars, magnetic, paramagnetic, conductive or insulating nanoparticles, synthetic particles, hydrogels (colloids or bars), and the like. The nanoparticles used in the present invention can exist as single nanoparticles, or as clusters or aggregates of the nanoparticles.
It will be appreciated by one of ordinary skill in the art that nanoparticles can exist in a variety of shapes, including but not limited to spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped nanoparticles, prism-shaped nanoparticles, and a plurality of other geometric and non-geometric shapes. Another class of nanoparticles that has been described includes those with internal surface area. These include hollow particles and porous or semi-porous particles. Moreover, it is understood that methods to prepare particles of these shapes, and in certain cases to prepare SERS-active particles of these shapes, have been described in the literature. While it is recognized that particle shape and aspect ratio can affect the physical, optical, and electronic characteristics of nanoparticles, the specific shape, aspect ratio, or presence/absence of internal surface area does not bear on the qualification of a particle as a nanoparticle.
A nanoparticle also includes a nanoparticle in which the metal includes an additional component, such as in a core-shell particle. For example, Ag core/Au shell particles, like those described in J. Am. Chem. Soc. 2001, 123, 7961, or Au core/Ag shell particles, or any core-shell combination involving SERS-active metals, can be used. Other combinations suitable for use in core-shell particles are included in this invention, such as Au- or Ag-nanoparticle functionalized silica/alumina colloids, Au- or Ag-functionalized TiO2 colloids, Au nanoparticle capped-Au nanoparticles (see, for example, Mucic, et al., J. Am. Chem. Soc. 1998, 120, 12674), Au nanoparticle-capped TiO2 colloids, particles having and Si core with a metal shell (“nanoshells”), such as silver-capped SiO2 colloids or gold-capped SiO2 colloids. (See, e.g. Jackson, et al., 2004 Proc Natl Acad Sci USA. 101(52):17930-5; Talley, et al., Nano Letters (2005)). Hollow nanoparticles such as hollow nanospheres and hollow nanocrystals may also be utilized in the SERS nanotags.
Each SERS nanotag is encoded with a unique reporter 16, comprising an organic or inorganic molecule at the interface between the nanoparticle core and shell of glass or other suitable encapsulant. This approach to detection tags leverages the strengths of Raman scattering as a high-resolution molecular spectroscopy tool and the enhancements associated with SERS, while bypassing the shortcomings often encountered when making stand alone SERS substrates such as difficult reproducibility and lack of selectivity. SERS nanotags exhibit intense spectra (enhancement factors in excess of 106) with the 633 and 785 nm excitation wavelengths that are excellent for avoiding intrinsic background fluorescence in biological samples such as whole blood and in matrices like glass and plastic. The glass coating, which is essentially SERS-inactive, stabilizes the particles against aggregation, prevents the reporter from diffusing away, prevents competitive adsorption of unwanted species, and provides an exceptionally well-established surface to which biomolecules can be conjugated for bioassay development (Aslam and Dent (1998) Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences. Grove's Dictionaries Inc, New York, N.Y.).
Multiple unique flavors of tags are available.
Certain embodiments of heterogeneous and homogeneous immunoassays consistent with the present invention feature derivatizing an inside wall of the test vial, with capture antibody. A whole blood or serum assay may be feasible with this format particularly if excess “unbound” tags are removed before Raman spectrum acquisition. It has been demonstrated under this format that SERS tags can be read in presence of neat plasma or whole blood and that excess tag removal is not required if the detection system provides for a suitably narrow depth of field.
Representative test vial wall based assays have been prepared according to the method detailed in Example 1 below. Other methods are equally suitable for preparing or performing an assay consistent with this aspect of the present invention.
A representative sequential (heterogeneous) test vial wall assay is schematically illustrated in
In the sequential assay, sample was added to the vial and incubated for 2.5 hours. Unbound analyte is washed off before incubation with the SERS nanotags for another two hours. Raman spectra, focusing on the bottom wall of the glass vial, were taken before removal of unbound SERS nanotags and again after washing away excess tags.
This assay format was used to screen antibody and select antibody pairs. After a set of mix-and-match experiments, a monoclonal anti-IL-2 antibody was chosen as the capture antibody and conjugated to derivatized vial. A polyclonal was chosen as the detection antibody.
Graphs 40 and 42 of
Raman spectra were collected for each IL-4 dose. The Raman spectra shown in
An alternative homogeneous assay format is schematically illustrated in
Chart 60 of
To demonstrate that high background is due to free tags in solution, a polylysine coated glass cover slip was loaded with a sub-monolayer of BPE tags. A suspension of a different tag (DPY) was added to the cover slip. Focusing on the surface the cover slip with a 50× objective (collection depth of ˜2 microns; Renishaw Raman Microscope), Raman spectra were collected at different time intervals.
Graph 70 of
When excess tags are washed off, background signal is dramatically reduced as shown in Graph 80 of
Before investigating the effect of a biological matrix on assay performance, it was demonstrated that the SERS nanotags can be read in the presence of plasma or blood.
The effect of human plasma and blood on assay performance has also been investigated. Plasma or blood concentrations at 30% of total assay volume negatively affected assay performance by giving high background signal and poor or no spike recovery—before and after wash. Graph 100 of
Representative method for the preparation of an assay featuring the capture of detection tags on an inside test vial wall.
Materials & Reagents
Buffers
Methods and Protocols
General Procedure for Antibody Conjugation to Dynabeads
Glass Vial Derivatization: APTMS
Succinic Anhydride Derivatization of APTMS Derivatized Vials
Antibody Conjugation to Thiolated-SERS Tags
Antibody Conjugation to APTMS and Carboxy-APTMS Coated Vials
Sequential IL-4 Assay Procedure—Vial Assay
9. Aspirate and read on Ocean Optics Reader.
Homogeneous IL-4 Assay Procedure—Vial
Certain embodiments of heterogeneous and homogeneous immunoassays consistent with the present invention feature the use of magnetic capture particles. The combined properties of magnetic capture particles and SERS nanotag detection particles results in a bioanalytical platform which avoids the use of robotics, extensive or complicated sample preparation and analytical separations, while permitting simple, quantitative, fast (real time) and multiplexed protein measurements.
Representative magnetic capture particle based assays have been prepared according to the method detailed in Example 2 below. Other methods are equally suitable for preparing or performing an assay consistent with this aspect of the present invention.
According to the method of Example 2, both capture and detection particles are loaded into a polypropylene tube (pre-diluted with the appropriate dilution buffer) followed by the sample. After incubation on an end-over-end mixing wheel, the tube is placed on a magnetic particle concentrator to pull down the magnetic particle to the bottom of the tube. The tube is then placed on the SERS reader and the laser is focused on the magnetic particles before recording the Raman spectrum.
A no-wash (homogeneous) sandwich immunoassay has been prepared for C-Reactive Protein using a magnetic particle derivatized with the capture antibody and a detection antibody conjugated to SERS nanotags as reporter as described above. Results indicate that the 2-particle complex can be analyzed in presence of biological matrices, without wash and using low cost detection systems.
The results of an IL-4 magnetic glass coated bead-based assay are illustrated by the dose-response curve 120 of
The above described one step, 30 minute, no-wash assay compares very favorably with the sensitivity (˜100 fg/ml) of the commercially available assays using the same antibodies but requiring multiple steps taking place over an entire day including multiple wash steps and 200 μl sample volume.
Numerous factors can impact assay sensitivity, including but not limited to:
Biocompatible Coating:
Reduction of non-specific particle—particle interaction can be achieved by judicious choice of biocompatible coatings which are usually selected from natural or synthetic polymers of different lengths, charges and polarity.
Antibody Concentration on SERS Nanotags:
A set of antibody SERS nanotag conjugates were prepared using 10 and 100-fold less antibody during the conjugation process. The resulting conjugates were tested and compared to non-reduced concentration conjugates. Both conjugates did not show activity most probably because of the overall reduction of detection antibody concentration in the system. However the minimum amount of antibody suitable for preparation of an effective assay has not yet been determined.
Assay Buffer Composition:
Buffer composition and buffer additives can have a dramatic effect on assay performance. Several qualities of assay blockers have been investigated. A preferred assay buffer for the CRP assay contains 5% BSA, 0.05% PEG, 0.05% Tween 20 in PBS. For an IL-4 assay a 1% BSA solution in PBS is suitable.
Capture and Detection Particle Concentration:
Multidimensional matrix experiments have been performed investigating primarily the effect of capture and detection particle concentration on assay performance. Graph 150 of
Assay sensitivity may be defined as the Limit of Detection calculated from the Mean value plus 2 standard deviations of 18 replicates of a zero calibrator (blank). Alternatively, 6 “zero calibrators” may be analyzed in parallel with proper control of timing. The following Table 1 gives the non-specific signal (obtained from multiple measurements of 6 replicates of a “blank” or “zero calibrator”. A coefficient of variance (cv) of ˜31% was determined, which is consistent with the average non-specific signal variability seen on a regular basis across independent experiments run.
This relatively high coefficient of variance shown in Table 1 indicates that more sufficient control of the non-specific interaction between the 2 particles is desirable, although reduction of their amounts appears to minimize the overall signal due to non-specific binding.
Assuming that this cv can be applied across all assays run, a Limit of Detection below 500 femtogram/ml may be determined for most of the dose-response curves. Here, the Limit of Detection was estimated from the Mean value plus 3 standard deviations of the zero calibrator (blank) since 6 replicates instead of 18 replicates was used to make the determination.
As described in detail below, an IL-4 assay may be carried out in presence of a biological matrix (for example a mid-range IL-4 spike may be recovered in 15% and 30% whole blood and serum) without further optimization. As illustrated in Graph 160 of
Assays prepared with magnetic capture particles and SERS nanotag detection particles are particularly well suited for the performance of multiplexed assays. In order to run a multiplexed dose response curve for combined IL-4 and CRP, unique detection tags for each analyte must be prepared. In addition, the 2 assays need to function in the same buffer (harmonized assay conditions). An example assay was prepared with a first BPE SERS nanotag. DPY was selected as a second tag and detection antibodies for both IL-4 and CRP were conjugated to both tags. Furthermore, harmonized buffer conditions need to be identified since the preferred assay buffer for CRP is PBS containing 5% BSA, 0.05% PEG and 0.05% Tween 20 whereas for IL-4 the preferred buffer is 1% BSA in PBS.
Graph 170 of
Graph 180 of
The following example of assays prepared with magnetic capture particles are provided for illustrative purposes only and are not intended to limit the scope of the invention. Applicant has performed experiments on an assay such as depicted in
1. Antibody Conjugation to Thiolated-SERS Nanotags
A solution of sulfo-SMCC (100 μL, 1 mg/ml in water) was added to solution of antibody (50 μg, 50 μl) in PBS. After 30 minutes, the maleimide-activated antibody was purified using a Sephadex G25 microspin desalting column (Amersham). The purified conjugate was then added to thiolated-SERS tags (4×1010 particles in 100 μL PBS). After 2 hours at room temperature, the reaction was quenched for 30 nm with a solution of MESA (50 μL, 10 mg/ml) followed by a 5% BSA solution (25 μL). The tag conjugates were washed, purified by centrifugation and resuspended in 0.1% BSA at the desired concentration in desired buffer.
2. Carboxylation of Magnetic Glass Coated Beads
Magnetic glass coated beads from Bioclone (4 mg) were washed 2 times with ethanol (1 ml) before treatment with a solution of 2% APTMS (3-aminopropyltrimethoxysilane) in ethanol (1 ml) for 30 nm at room temperature. The beads were washed via magnetic pulldown and treated with ethanol (500 μl) on a hot plate (50 C) for 30 nm. After washing with ethanol and water (3 times), the magnetic particles were stored in water at 4 mg/ml. Carboxylation was done in borate buffer by treatment with a succinic anhydride solution in DMSO (20 μl, 40 mg/ml) in DMSO for 30 nm. The carboxylated beads were washed and stored in water.
3. General Procedure for Antibody Conjugation to Carboxylated Magnetic Glass Coated Beads and Dynabeads
Carboxylated magnetic glass coated beads or Dynabeads M-270 carboxylic acid (1 mg) in PBS (100 μl) were treated with antibody (30 μl 1 mg/ml) for 30 minutes before addition of an EDC solution (10 μl, 1 mg). The mixture was incubated with slow tilt rotation at room temperature for 2 hours before blocking for 0.5 hour with ethanolamine in PBS (200 μl, 3 mg/ml). The conjugate was then washed and further blocked overnight with a solution of 1% BSA in PBS and stored in the same buffer at 4 C.
4. Homogeneous Magnetic Beads Based Assay for C-Reactive Protein (CRP)
An Eppendorf tube (polypropylene, 500 μl) was sequentially loaded with the following reagents
The mixture was incubated at room temperature by gentle mixing for 30 nm on mixing wheel. The magnetic particles were pulled to the bottom of the tube using a magnetic particle concentrator. The tubes were placed on the Raman reader and adjusted to position the bottom of the tube (i.e. magnetic beads) into the focal plane of the optical detection system. After a 1s reading time, the acquired data was reduced with a proprietary software package that allows analyte concentration determination.
Data was collected using 1 um glass coated magnetic particles from Dynal, and bis(4-pyridyl)ethylene (BPE) SERS nanotags to detect CRP in buffer. The Raman spectra were collected on an Inspector Raman instrument from Delta Nu Corporation. The Inspector Raman system employs 40 mW, 785 nm laser excitation, free space optics, is portable and can be interfaced to a laptop computer. Data was analyzed using a software program performing least squares fitting of spectra to quantify components in complex mixtures. Graph 210 of
The dose-response in buffer described above was repeated several times with good assay reproducibility by giving standard deviation with coefficient of variation (cv) below 15% for triplicate reading of the same tube. The blank was read 20 times to give a cv of 20% and a intra-assay Limit of Detection of 200 pg/ml. This detection limit is more than 500 fold lower than the average normal physiological concentration of human CRP (100 ng/ml).
SERS nanotags can be read in a background of biological matrix, such as plasma or blood.
Not all conjugated antibodies remain active and not all particles remain colloidally stable after conjugation. Multiple attachment chemistries, multiple types of magnetic particles (differing by size, composition and surface functionality), multiple SERS tags functionalities and multiple antibodies for each analyte may be investigated.
It is important to develop robust and controllable methods of bioconjugation to both the capture and detection particle. Since suitable SERS nanotags and magnetic particles may be glass encapsulated it may be possible to use the same bioconjugation chemistries. The surface silanol groups can be easily derivatized with commercially available carboxy-, mercapto-, and aldehyde-silanes allowing a large optimization space to be explored. The preferred protocol for surface modification relies on thiolated SERS nanotags. Carboxylates, amines and aldehydes functionalities could give more stable intermediates. Different attachment chemistries (corresponding to different reaction conditions) may be investigated. This should cover buffer requirement (Pi) for most biological molecules and avoid potential for aggregation and denaturation during conjugation, as shown in Table 2 (Kricka (2005) Microchips, microarrays, biochips and nanochips—personal laboratories for the 21st century. http://www.ifcc.org/ejifcc/vol12no4/vol12no4a2.htm; MacBeath and Schreiber (2000) Science 289:1760-1763; Kusnezow and Hoheisel (2003) J. Molecular Recognition 16:165-176; Nemzek et al. (2001) J. Immunol. Methods 255:149-157). For example, reductive amination offers several advantages since it works over a wider pH range than the carbodiimide reaction (EDC), and therefore the pH can be controlled and adapted to the antibody stability/buffer requirement (Hermanson (1996) Bioconjugate Techniques. Academic Press). Antibody precipitation or particle aggregation could be avoided in this manner. Other advantages are that the amine bond is more stable than an amide bond, the overall charge of the antibody remains after reductive amination and there is no occurrence of antibody cross-linking as is very often observed during the carbodiimide reaction.
The major variable for is in the elimination of non-specific binding (NSB) and in an understanding of the interaction between the magnetic particle and SERS nanotag. The level to which each of the different conjugation protocols, and surface coverage, affect NSB may be investigated. The use of standard blocking agents and wash buffers (for example BSA, Tween, and casein, in addition to different pH and buffers) may be investigated. The time of reaction and concentration of particles will also be critical. It will be important that particles do not aggregate non-specifically. It is expected that non-specific binding will be minimized when the magnetic particle and SERS nanotag have the same surface characteristics. Should NSB prove insurmountable, polymer based derivatization of the SERS nanotag glass shell, such as polyacrylamide, polyethylene glycol and dextrans, polyethylenimine will be investigated (Lee et al. (2002) Bioconj. Chem. 13:97-103) and dendrimers (Le Berre et al. (2003) Nucleic Acids Res. 31:e88), all known to have low NSB. The optimized protocols of biofunctionalization of the SERS nanotags and magnetic particles by assessment of their pair-wise assay performance; determining sensitivity, dynamic range and specificity in buffer may be selected.
Glass-coated, superparamagnetic particles may be used as capture particles. Other particles commonly utilized for magnetic separation in molecular biology and readily available through companies such as Dynal, Bangs Laboratories, CPG Inc., and PerSeptive Biosystems may also be considered. These typical magnetizable particles have been extensively used in enzyme immunoassay (8) and for other binding assays in a wide size range (50 nm to several micrometers). A 2.8 micron carboxylated polystyrene coated magnetic particle from Dynal has been compared with a 1 micron glass coated magnetic particle from Bioclone (Bioclone Magnetic Particles (2005) http://www.bioclon.com/functional-magnetic-paramagnetic-NH2—COOH-silica-epoxy-IDA-beads.html) and it has been found that the less porous glass-coated particles gave lower non-specific interaction of SERS nanotags in the CRP dose-response data. Particles can be obtained with different surface functional groups such as amine, carboxy, aldehyde, epoxy, silica, hydrazide, and others that have been used for stable and covalent attachment of antibodies.
Spectrally distinct SERS nanotags are manufactured in a highly reproducible manner (examples are illustrated in
The data discussed above was obtained in an eppendorf tube made of polypropylene. A major advantage of Raman analysis is that many materials of assay vessels used in the bioassay community are transparent to Raman light scatter, and the background Raman signal of these materials is in general minimal. The many proposed assay formats are not limited by the nature of the assay vessel (as is very often the case with fluorescence) but more importantly, the SERS signal can be read through the wall material of many assay vessels. The only limitation is that the container wall does not interfere/absorb the laser light energy (typically 650 or 783 nm). A wide variety of inexpensive and commonly used assay vessels, including 96-well plates, tubes, vials, vacutainers and cups may be suitable for the described assays. The following material types: Glass, silica, silicon, silanized glass, polypropylene, polyethylene, polyurethane, Teflon, polystyrene, nitrocellulose, cellulose, polyester, and polycarbonate may be suitable for use in assay vessels.
SERS nanotags and magnetic beads may be hybridized to function as a SERS magnetic DNA assay.
An advantage of a biotin:streptavidin hybridization approach is that the target sequence being interrogated does not need to be labeled with SERS tag, but may be labeled with biotin instead. However, multiplexing is difficult. It may be desirable to employ a three-piece ligation to allow multiplexing. Alternatively, the conjugation order may be reversed with the original sequences first attached to the different “flavors” of SERS nanotags, followed by streptavidin coating of the magnetic particles.
Graph 2300 of
In tube 1 N30A target was added to magnetic beads conjugated to N30A sequence (shown on the left of the N30A designated results of
Graph 2400 of
Hybridization
A homogenous two particle competitive assay consistent with the present invention may also be implemented with the use of SERS Nanotag detection particles and a separate capture particle. Certain advantages described below are realized if the capture particles are magnetic, but the scope of this aspect of the invention is not limited to magnetic capture. A competitive assay format may be selected to implement any type of bioassay diagnostic test. The competitive format is particularly advantageous however, for the detection of analytes which are too small to accommodate two different antibodies capable of forming a sandwich type immune complex as described above. A competitive assay format as described herein may be utilized to detect any type of analyte including but not limited to therapeutic drugs, non-therapeutic drugs (drug abuse monitoring), peptides, hormones, proteins, or other analytes of interest. The competitive assay format is also particularly advantageous where only one binding agent is readily available for the analyte of interest.
Certain specific competitive assay formats featuring capture particles and SERS nanotag detection particles are described in detail below. In general, however, all competitive assay formats require the labeling of either the capture particle or the detection particle with the analyte or an analog thereof. The detection particle may be optically labeled such as fluorescent, colored or SERS active particle. The particle not labeled with the analyte or an analog thereof is conjugated to a binding agent suitable for binding the analyte. For example, as is shown in
The capture particles 2500 may be magnetic capture particles or other particles which are suitable for concentration. In one non-magnetic embodiment, the capture particles are large or dense particles pulled down by centrifugation. Large and/or dense capture particles include, but are not limited to, Nanobarcodes® Particles, spherical or anisotropic metallic nanoparticles, or any type of particle consisting of, but not limited to, latex, polystyrene, or other material, with sufficient specific gravity, size, and shape to be pulled down by centrifugation in the solvent of interest. In the
Alternatively, as shown in
In a first competitive format, the capture and detection particles are added in rapid sequence to the analyte containing sample. During the incubation, both free analyte or an analog of the analyte either of which is referred to as “A” and particle-conjugated analyte may compete for conjugated binding agent “R”. The binding agent “R” may have a different affinity for an analog A (preferably lower) compared to the affinity for endogenous A. After incubation, the magnetic particles (including free MP particles and MP-R::A-SERS or SERS-R::A-MP complexes depending upon which particle was conjugated with the binding agent) may be concentrated at a determined location within the reaction vessel followed by Raman spectrum acquisition. The Raman signal intensity is inversely proportional to the concentration (amount) of free (endogenous) analyte present in the sample. The possible complexes formed in this competitive assay, depending on which particle is derivatized with the analyte or analog are:
MP-A+A+SERS-R→SERS-R::A+SERS-R::A-MP; OR
SERS-A+MP-R+A→MP-R::A+MP-R+SERS-A+MP-R::A-SERS
In a second competitive assay format, the analyte containing sample is first incubated with the particle conjugated to the binding agent “R” (MP-R or SERS-R) for a given time to allow for capture of endogeneous analyte “A” before addition of the particle conjugated to A. The later added analyte-derivatized particle then binds to the residual particle-R conjugate (MP-R or SERS-R). After incubation, the magnetic particles (free MP particles and MP-R::A-SERS or SERS-R::A-MP complexes) are concentrated at a determined location within the reaction vessel followed by Raman spectrum acquisition. The spectroscopically determined number of MP-R::A-SERS or SERS-R::A-MP is inversely proportional to the concentration of free (endogeneous A) present in the sample. The two step pseudo-competitive process includes the following reactions, depending upon which particle is conjugated with the binding agent.
SERS-R+A→SERS-R::A+SERS-R(SERS-R::A+SERS-R)+MP-A→SERS-R::A+SERS-R::A-MP; OR
MP-R+A→MP-R::A+MP-R(MP-R::A+MP-R)+SERS-A→MP-R::A+MP-R::A-SERS
In yet another assay format, SERS-R::A-MP (or MP-R::A-SERS) may be pre-formed and stored in the assay vessel in either liquid or lyophilized form. Upon the addition of sample, this particle-particle complex dissociates by interaction with endogeneous A in proportion to the analyte concentration, according to the following reactions.
MP-R::A-SERS+A→MP-R::A+MP-R::A-SERS; OR
SERS-R::A-MP+A→SERS-R::A+SERS-R::A-MP
SERS Nanotag detection particles and separate capture particles may also be used to implement varied types of assays for the detection of nucleotide sequences. The nucleotide sequences which are suitable for detection by an assay as described herein may be from any source or organism. For example, nucleotide sources include but are not limited to human, animal, plant, bacteria, viral or other genetic material. Certain advantages as described below are realized if the capture particles are magnetic but the scope of this aspect of the invention is not limited to embodiments featuring magnetic capture particles. The nucleotide sequences detection assays described herein allow for the detection of target nucleotide sequences in crude lysates and impure DNA preparations without the need for washing steps or the removal of sample mixtures. Certain assays described herein can be completed in a short time, for example, a half hour or less.
One nucleotide sequences detection assay consistent with the present invention is schematically illustrated in
The assay illustrated on
After incubation of the hybridized probes with the SERS nanotag complex, the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag may be detected with a Raman spectrometer, Raman microscope or other detection device 2618. Concentration or selection of the resulting hybridized complex must be accomplished to separate hybridized nanotags from unbound nanotags. If a magnetic particle 2604 was used to prepare a capture probe, selection of the magnetic particles may be accomplished using known magnetic concentration techniques.
The data of experimental validation of the effectiveness of the
An alternative nucleotide sequences detection assay is schematically illustrated in
Graph 3400 of
One method of increasing nucleotide assay sensitivity is schematically illustrated in
Alternatively, a larger nucleotide fragment (for example, a genomic or gene fragment without digestion or with minimal digestion) can be hybridized with a capture sequence conjugated to a capture particle. Furthermore, multiple different detection sequences complementary to the large nucleotide fragment may each be conjugated with same SERS nanotag. Assay sensitivity will thus increase by increasing the number of detection probes on a single larger nucleotide sequence. In this case the assay specificity could also be increased by using a plurality of SERS nanotags. Alternatively, if multiple different detection sequences have been conjugated with different SERS nanotags, or a specific genomic deletion or mutation can be identified by designing specific detection sequences to detect the region of interest. Furthermore, the magnetic particle could be conjugated to multiple different capture sequences complementary to different nucleotide fragments of interest resulting in the assay signal being enhanced for each magnetic particle.
Other methods may be used to increase nucleic acid assay sensitivity. For example, isothermal rolling circle amplification (RCA), polymerase chain reaction (PCR) related amplification, T7 RNA polymerase-driven amplification, ligase chain reaction (LCR), branched DNA (bDNA) or other known amplification methods may be used in conjunction with capture and detection probes as described above. An example assay featuring RCA is illustrated in
Carboxyl Functionalization:
Conjugation of Oligo to Functionalized SERS Tags
Conjugation of Oligo to Magnetic Beads
Hybridization
3. Add 1× NeutrAvidin-BPE (SERS tag) and incubate for 30 minutes.
SERS Rolling Cicle Amplification:
The various embodiments disclosed herein include but are not limited to the following:
A method of detecting a nucleotide of interest comprising:
providing a capture probe comprising a capture sequence conjugated to a magnetic particle; providing a detection probe comprising a detection sequence conjugated to a capture molecule; providing a SERS nanotag conjugated to a complementary target capable of selectively binding the capture molecule; hybridizing the capture probe and detection probe in the presence of the nucleotide of interest; contacting the hybridized capture and detection probe with a the SERS nanotag complex; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag.
The above method of detecting a nucleotide of interest further comprising magnetically selecting the magnetic particle.
A method of detecting a nucleotide of interest comprising:
providing a capture probe comprising a capture sequence conjugated to a magnetic particle; providing a detection probe comprising a detection sequence conjugated to a SERS nanotag; hybridizing the capture probe and detection probe in the presence of the nucleotide of interest; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag.
The above method of detecting a nucleotide of interest further comprising magnetically selecting the magnetic particle.
A method of detecting a nucleotide of interest comprising:
cutting the nucleotide of interest into multiple sections; providing multiple capture probes having distinct hybridization affinities for separate portions of the nucleotide of interest, the multiple capture probes being comprised of capture sequences conjugated to magnetic particles; providing multiple detection probes having distinct hybridization affinities for separate portions of the nucleotide of interest, the detection probes being comprised of detection sequences conjugated to SERS nanotags; hybridizing the capture probes and detection probes in the presence of the cut nucleotide of interest; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotags.
The above method of detecting a nucleotide of interest wherein the nucleotide is cut into multiple sections with a restriction enzyme.
The above method of detecting a nucleotide of interest further comprising magnetically selecting the magnetic particles.
A method of detecting a nucleotide of interest comprising:
providing a capture probe comprising a capture sequence conjugated to a magnetic particle; providing an RCA probe; hybridizing the capture probe and RCA probe in the presence of the nucleotide of interest; performing rolling circle amplification on the initial hybridization products; providing a detection probe comprising a detection sequence conjugated to a SERS nanotag; incubating the initial hybridization products in the presence of the detection probe; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag.
The above method of detecting a nucleotide of interest further comprising magnetically selecting the magnetic particle after performing rolling circle amplification on the initial hybridization products.
The above method of detecting a nucleotide of interest further comprising magnetically selecting the magnetic particle after incubating the initial hybridization products in the presence of the detection probe.
The above method of detecting a nucleotide of interest further comprising magnetically selecting the magnetic particle after incubating the initial hybridization products in the presence of the detection probe.
An assay for detecting a nucleotide comprising:
a capture probe comprising a capture sequence conjugated to a magnetic particle; a detection probe comprising a detection sequence conjugated to a capture molecule; and a SERS nanotag conjugated to a complementary target capable of selectively binding the capture molecule.
An assay for detecting a nucleotide comprising:
a capture probe comprising a capture sequence conjugated to a magnetic particle; and a detection probe comprising a detection sequence conjugated to a SERS nanotag.
An assay for detecting a nucleotide comprising:
means for cutting a nucleotide of interest into multiple sections; multiple capture probes having distinct hybridization affinities for separate portions of the nucleotide of interest, the multiple capture probes being comprised of capture sequences conjugated to magnetic particles; and multiple detection probes having distinct hybridization affinities for separate portions of the nucleotide of interest, the detection probes being comprised of detection sequences conjugated to SERS nanotags.
A method of detecting a molecule of interest comprising:
providing a magnetic bead conjugated to a molecule; hybridizing the magnetic bead with a complementary target capable of selectively binding the molecule, the target being further associated with a detection moiety; contacting the hybridized magnetic bead with a SERS nanotag capable of binding the detection moiety; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag.
The above method wherein the target is a nucleic acid molecule.
A method of detecting a molecule of interest comprising:
providing a SERS nanotag conjugated to a molecule; hybridizing the SERS nanotag with a complementary target capable of selectively binding the molecule, the target being further associated with a detection moiety; contacting the hybridized SERS nanotag with a magnetic particle capable of binding the detection moiety; magnetically selecting the magnetic particle; and detecting a Raman spectrum associated with the SERS nanotag.
The above method wherein the target is a nucleic acid molecule.
A method of detecting a molecule of interest comprising:
providing a first SERS nanotag conjugated to a first molecule; providing a second SERS nanotag conjugated to a second molecule wherein the second SERS nanotag presents a different Raman spectrum from the first SERS nanotag upon interrogation; hybridized the first and second SERS nanotags with a complementary target capable of selectively binding one of the first and second molecules, the target being further associated with a detection moiety; contacting the hybridized first and second SERS nanotags with a magnetic particle capable of binding the detection moiety; magnetically selecting the magnetic particle; and detecting the Raman spectrum of one of the first and the second SERS nanotag.
The above method wherein the first and second molecules are nucleic acid molecules.
A method for detecting an analyte of interest comprising:
providing capture particles conjugated with a first molecule capable of selectively binding said analyte; providing SERS nanotag detection particles conjugated with a second molecule capable of selectively binding said analyte; contacting a sample which may contain the analyte of interest with the capture and detection particles, thereby forming a 2-particle complex from capture particles and detection particles bound to the analyte; concentrating the 2 particle complex; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag.
The above method wherein the capture particles are magnetic particles.
The above method wherein the concentration step is performed on a magnetic particle concentrator.
The above method wherein the 2 particle complex is concentrated on the interior wall of a test vessel.
The above method wherein the 2 particle complex is captured on the interior wall of a test vessel.
A method of detecting an analyte of interest comprising:
providing capture particles derivatized with said analyte; providing SERS nanotag detection particles conjugated with a first molecule capable of selectively binding said analyte; contacting a sample which may contain the analyte of interest with the capture and SERS nanotag detection particles, thereby forming both an analyte/SERS nanotag detection particle complex and a capture particle/SERS nanotag detection particle complex; concentrating the capture particle/SERS nanotag detection particle complex; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag detection particles.
The above method wherein the capture particles are magnetic particles.
The above method wherein the concentration step is performed on a magnetic particle concentrator.
A method for detecting an analyte of interest comprising:
providing capture particles conjugated with a first molecule capable of selectively binding said analyte; providing SERS nanotag detection particles conjugated with a second molecule capable of selectively binding said analyte; contacting a sample which may contain the analyte of interest with the capture and detection particles, thereby forming a 2-particle complex from capture particles and detection particles bound to the analyte; concentrating the 2 particle complex; and detecting the Raman spectrum of a Raman reporter molecule associated with the SERS nanotag.
The above method wherein the capture particles are magnetic particles.
The above method wherein the concentration step is performed on a magnetic particle concentrator.
The above method wherein the 2 particle complex is concentrated on the interior wall of a test vessel.
While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention.
This application claims priority to and is a continuation application of PCT/US2007/061878, currently pending, filed Feb. 8, 2007, entitled “SERS Nanotag Assays,” which international patent application claims the benefit of priority to: U.S. Provisional Patent Application No. 60/771,766, filed Feb. 8, 2006, entitled “SERS Nanotag Biological Assays;” U.S. Provisional Patent Application No. 60/832,917, filed Jul. 24, 2006, entitled “SERS Nanotag Biological Assays;” and U.S. Provisional Patent Application No. 60/870,963, filed Dec. 20, 2006, entitled “SERS Nanotag Biological Assays,” which are each incorporated herein in their entirety by reference for all that they teach and disclose.
| Number | Date | Country | |
|---|---|---|---|
| 60771766 | Feb 2006 | US | |
| 60832917 | Jul 2006 | US | |
| 60870963 | Dec 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12184691 | Aug 2008 | US |
| Child | 13658542 | US | |
| Parent | PCT/US2007/061878 | Feb 2007 | US |
| Child | 12184691 | US |
