This application claims the benefit of Korean Patent Application No. 10-2013-0038147, filed on Apr. 8, 2013, the contents of which is incorporated herein by reference.
This invention relates to a target-specific probe comprising a ferritin protein and a targeting agent, and a detection method, a quantification method and a detection kit of a biomarker using the probe.
A biomarker is a type of biomaterials present in biological or medical specimens, which functions as a marker capable of diagnosing the condition of a disease by detecting a change in the structure or concentration thereof qualitatively and/or quantitatively and determining the treatment effects of a medicine and correlation with other diseases comprehensively. For the early diagnosis of diseases, it is essential to analyze a biomarker occurring in the beginning stage of the diseases quantitatively. However, technologies currently available for monitoring the diseases do not properly meet technical needs for the early diagnosis of diseases because of limits such as sensitivity, quarantine speed, and costs.
ELISA (Enzyme-Linked ImmunoSorbent Assay), western blotting, and a mass spectrometry-based method have been generally used to quantitatively analyze biomarkers. In case of ELISA, accurate detection is difficult because a reaction may be inhibited by polysaccharides or phenol compounds present in the test samples or the concentration of bacteriophages present in tissues is low. While the mass spectrometry-based method has very good sensitivity so that they are applicable to analyze a slight amount of a biomarker, it has difficulty in securing reproducibility because this is usually analyzed by being linked to chromatography method and it also has a huge deviation of analysis data due to machine errors. In addition, these methods require excessive labor and a large amount of time.
Recently, according to the rapid development of nanotechnologies, detection technologies which were impossible under the previous detection methods are emerging. For example, Lieber et. al from Harvard University published a nano-detecting sensor for detecting a single bacteriophage (Science, vol. 329, pp. 830-4, Aug. 13 2010), and Mirkin et. al from Northwestern University established Nanosphere company, on the basis of a molecule detection technology using a nanoprobe (Sensors, vol. 12, pp. 1657-1687, Feb. 7 2012).
However, while the methods based on nanoparticles have excellent detection sensitivity, two-dimensional detection methods such as nano elements have poor accessibility toward a subject to be detected to quantitatively analyze minute amounts (
A quantum dot is an inorganic semiconductive substance having a nano size, which is recently applied to various medical engineering fields because of its excellent optical properties including high quantum efficiency, excellent resistance to photo fading, the control of fluorescence property by size, and non-overlapped fluorescence spectrum. Attempts to use quantum dots have been made for the quantification of important disease markers (Analytical Chemistry, vol. 76, pp. 4806-4810, Aug. 15 2004; Analytical Chemistry, vol. 82, pp. 5591-5597, Jul. 1 2010).
Furthermore, in order to overcome quenching phenomenon where the fluorescence intensity of quantum dots becomes dramatically weak, attempts to measure the number of quantum dots in a different manner were published. For example, the change of electrical conductivity due to cadmium ions which constitute quantum dots was measured by dissolving the quantum dots in a strong acid when Prostate-specific Antigen (PSA) was separated, detected, and quantified (Small, vol. 4, pp. 82-86, January 2008), but there was an issue that a large amount of cadmium ions which are toxic substances were generated. In another analysis example using quantum dots, with the purpose of measuring intrinsic fluorescence by the separation of quantum dots, organic solvents and alkali solutions having a high concentration were used to cleave Streptavidin-Biotin bond (Analyst, vol. 135, pp. 381-389, 2010.), but there were issues that the aggregation of quantum dots might occur due to the organic solvents, various buffering solutions were required because of the deterioration of the stability and optical properties of the quantum dots under experiment conditions, and it had poor reproducibility.
Therefore, in order to detect biomarkers with high sensitivity, a new detection system of biomarkers capable of overcoming the limits of the previous detection systems including nanoparticles and nano elements, being simply analyzed, being easily handled, and providing accurate detection results with high sensitivity is needed.
It is an object of the invention to provide a target-specific probe comprising a ferritin protein and a targeting agent.
It is another object of the invention to provide a detection method, a quantification method and a detection kit of a biomarker using the target-specific probe, capable of overcoming the limits of the previous detection systems including nanoparticles and nano elements, being simply analyzed, being easily handled, and providing accurate detection results with high sensitivity.
In order to achieve the above-mentioned objects, there is provided in one embodiment of the present invention a target-specific probe comprising a ferritin complex comprising a ferritin protein and a coupling partner coupled to the ferritin protein, and a targeting agent coupled to the ferritin complex.
Preferably, the ferritin complex may comprise a peptide or protein, or a compound as a coupling partner, and when the coupling partner is a peptide or protein, the target-specific probe comprises the ferritin protein; and the peptide or protein which is connected to the C-terminal or N-terminal of the ferritin protein and is a fusion partner not inhibiting ferritin structure formation. When the coupling partner is a compound, the ferritin and the compound are coupled to form a ferritin complex, which is then coupled to a targeting agent to form a target-specific probe.
Another embodiment of the invention relates to a method for detecting a biomarker comprising contacting a target-specific probe comprising a targeting agent specific to the biomarker and a detectable labeling agent to a sample containing the target biomarker to be detected, and detecting the labeling agent of the biomarker-targeted probe.
In the method for detecting a biomarker, when the labeling agent is detected, the biomarker can be determined to be present in the sample, or the amount of the biomarker in the sample can be measured by producing a standard curve of the labeling agent and comparing the detectable amount of the biomarker-targeted labeling agent with the standard curve.
Further embodiment of the present invention relates to a detection kit comprising a target-specific probe and a detector for detecting a labeling agent of the targeted probe.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Hereafter, the present invention will be described in more detail.
The probe according to the invention is a target-specific probe comprising a ferritin complex of a ferritin protein-a coupling partner; and a targeting agent coupled to the ferritin complex.
In this specification, the targeting agent is a targeting agent having a targeting ability specific to a biomarker which is a subject to be detected or measured and for example, it may be an antibody, aptamer, aptide, or peptide. The antibody may be a monoclonal antibody or polyclonal antibody, an immunologically active fragment (e.g., Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, a gene-engineered single chain molecule, a chimeric antibody, or a humanized antibody. The coupling of the ferritin fusion protein with the targeting agent enables the target-specific probe to move toward the biomarker target to be detected.
In the specification, the ferritin protein refers to comprise the ferritin protein itself, a heavy chain of ferritin, a light chain of ferritin, an analogue thereof, and apoferritin. Ferritin is a protein aggregate widely present in extracellular matrix, which consists of 24 single subunits and forms a cage-like nanostructure of which the outer diameter is 12 nm and the inner diameter is 8 nm. The ferritin cage has an internal empty space having the size of about 8 nm, contains in its inside about 4500 Fe atoms in their ferric oxide state, and serves to supply such Fe atoms during metabolic process.
The ferritin complex may be either in the form of a fusion protein produced by the coupling of the ferritin protein and a peptide or protein, or in the coupled form of the ferritin protein and a compound. As the coupling partners applicable to the present invention, any desired substances including Protein G, Fc receptor, Protein A, Protein A/G, Biotin, Avidin, and Streptavidin can be used.
In an aspect of the invention, when the ferritin complex is a fusion protein produced by bonding the ferritin protein and a peptide or protein, the ferritin complex may be a ferritin fusion complex comprising the ferritin protein; and the protein or peptide which is connected to the C-terminal or N-terminal of the ferritin protein and is a fusion partner not inhibiting ferritin structure formation.
The fusion partner and the ferritin protein may be directly connected, or fused using a linker peptide consisting of 30 or less amino acids, preferably, 15 to 25 amino acids. The coupling partner of the ferritin fusion protein aids the ferritin complex in functioning to be targeted at the biomarker by being coupled with the targeting agent. The coupling partner capable of being expressed as the ferritin fusion protein may include Protein G, Fc receptor, Protein A, or Protein A/G, but not be limited thereto.
When the targeting agent is an antibody, the targeting ability can be maximized by using the property that the coupling partner specifically binds to the Fc portion of the antibody targeting agent so that the Fab portion of the biomarker-targeted antibody can be always activated. In chemical binding methods using N-hydroxysuccinimide (NHS), and ethyl(dimethyl aminopropyl) carbodiimide (EDC) (Nat Protoc 2007, 2 (5), 1152-1165), there is a high possibility that the Fc portion of an antibody may not be activated due to a non-specific binding between the antibody and nanoparticles, eventually decreasing the targeting efficiency. Hence, in order to overcome such a low efficiency, an expensive antibody needs to be used for reaction in excessive amounts.
The target-specific probe according to an aspect of the invention may further comprise a detectable labeling agent which is coupled to the ferritin complex. The present invention relates to a target-specific imaging probe comprising one or two more detectable labeling agents which are coupled to a linker for coupling the labeling agents of the target-specific probe. The target-specific imaging probe may further comprise a linker for coupling the detectable labeling agent, selected from HIS tag, CYS tag, GST tag, Biotin tag, Avidin tag, and Streptavidin tag connected to the terminal of the ferritin protein or the coupling partner. Preferably, the labeling agent may be coupled to the ferritin complex via the linker, and the subject biomarker-targeted probe may be detected using the labeling agent.
The labeling agent applicable to the invention may include a quantum dot, magnetic bead nanoparticle, gold nanoparticle, fluorescent dye, fluorescent protein, nano phosphor, or silicon nanoparticle, and the labeling agent may be detected by fluorescence microscopy, SEM, TEM, CT, MRI, etc.
The labeling agent in itself may be coupled to a linker for coupling the labeling agent, selected from HIS tag, CYS tag, GST tag, Biotin tag, Avidin tag, and Streptavidin tag, or it may be coupled to the linker after the chemical treatment to the labeling agent to increase a binding ability toward the linker.
As a method for coupling the labeling agent to the ferritin complex, for example, a polymerase chain reaction (PCR) may be utilized. A desired labeling agent can be synthesized by performing PCR, using a fusion protein as a template and including the labeling agent in primers. Alternately, it can be performed by using a fusion protein as a template, inserting two desired restriction enzyme sites into the fusion protein, likewise inserting the same kinds of restriction enzyme sites into the both ends of the labeling agent, cleaving the genes with the restriction enzymes, and then fusing the cleaved genes using ligation.
When the ferritin complex is a compound coupled to ferritin, the labeling agent binds to a correspondent compound capable of binding to the compound, thereby forming a bond between the compound of the ferritin complex and the correspondent compound of the labeling agent to prepare a target-specific probe containing the detectable labeling agent. In particular, the ferritin complex may be a complex formed by coupling Biotin tag, Avidin tag, or Streptavidin tag compound as the coupling partner to the ferritin protein, and the invention may be a target-specific probe comprising a compound coupled to the ferritin complex, and capable of chemically binding to the compound coupling partner as a detectable labeling agent. For example, when Biotin is the coupling partner, Streptavidin can be used as the labeling agent.
While the detectable labeling agent may be coupled in 1 to 24 moles per mole of the ferritin complex, 2 to 15 moles are preferable in consideration of the high detection rate and sensitivity of the labeling agent, and space between the labeling agents.
The labeling agent may be a quantum dot, magnetic bead nanoparticle, gold nanoparticle, fluorescent dye, fluorescent protein, nano phosphor, or silicon nanoparticle.
When the labeling agent is a quantum dot, it may be used in itself, or it may be a quantum dot comprising a hydrophilic surface layer thereon obtained by treating the surface with an amphiphilic substance containing both of hydrophilic group and hydrophobic group. Particularly, it is preferable that the quantum dots show the least aggregation phenomenon by possessing hydrophilicity and it is more preferable that they are functionalized with nickel. For example, the hydrophilic surface may be obtained by treating it with one or more substances selected from the group consisting of 1-Myristoyl-2-Hydroxy-sn-Glycero-3-Phosphocholine (MHPC), 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-en-Methoxypolyethyleneglycol-2000 (DPPE-PEG2000), and 1,2-Dioleoyl-sn-Glycero-3-en-{5-amino-1-carboxylpentyl}iminodiacetic acid succinyl nickel salt (Ni-NTA). The amphiphilic substances may be treated alone or in a combination of two or more.
In particular, MHPC can be densely coated onto the surface of a sphere since it is a single acyl group chain lipid, DPPE-PEG2000 can confer stability on the quantum dots coated with the lipids, and Ni-NTA can bind to histidine on ferritin to bond hydrophilic quantum dots and ferritin. Thus, a functionalized probe capable of quantifying a biomarker with super sensitivity can be prepared by adding quantum dots having hydrophilic surface to the biomarker-targeted antibody and ferritin complex,
According to a specific embodiment of the invention, MHPC, DPPE-PEG2000, and Ni-NTA may be used alone or preferably, they can be used in a mixture form comprising the three components. For example, 50 to 95 mole % of MHPC, 4 to 35 mole % of DPPE-PEG2000, and 1 to 15 mole % of Ni-NTA are mixed with quantum dots to become an emulsion state, which is then sonicated to form a hydrophilic surface layer on the surface of the quantum dots. The above composition is merely mentioned as one illustration, and the number and the ratio of the lipids to be used may be adjusted to the desired purpose.
A preferred target-specific probe according to the invention may be formed by the sequential binding of protein G or Fc receptor, and ferritin from its N-terminal to C-terminal, wherein the Fc portion of the targeting antibody agent may be connected to protein G or Fc receptor of the ferritin fusion protein and more preferably, it may comprise HIS tag, CYS tag, GST tag, Biotin tag, Avidin tag, or Streptavidin tag which is connected to the terminal of the ferritin protein or the terminal of the coupling partner.
Another aspect of the invention relates to a method for detecting a biomarker comprising contacting the target-specific probe containing a targeting agent specific to the biomarker and a labeling agent to a sample containing the biomarker, and detecting the labeling agent of the biomarker-targeted probe.
Also, another aspect of the invention relates to a detection kit of a biomarker comprising a target-specific probe and a detector for detecting a labeling agent of the targeted probe. The target-specific probe has been described in detail in the above.
The method for detecting biomarker may further comprise a step in which the biomarker is determined to be present in the sample when the labeling agent is detected. Furthermore, it may be a method in which a standard curve is produced with detectable amounts measured at various concentrations of the labeling agent, and the amount of the biomarker in the sample is measured by comparing the detectable amount of the biomarker-targeted labeling agent with the standard curve.
As a specific example of the invention,
According to the invention, super sensitivity quantity analysis is carried out by measuring the fluorescence intensity values of quantum dots after targeting a biomarker at the surface of a cell/tissue using ferritin and quantum dots, and a detection kit may be manufactured using this. Accordingly, the biomarker detection kit comprises a target-specific probe and a detector for detecting a targeted probe and labeling agent.
The present invention provides a target-specific probe containing a ferritin fusion protein and a targeting agent, as a new detection system of a biomarker capable of being simply analyzed, being easily handled, and providing accurate detection results with high sensitivity, and a detection method and detection kit of a biomarker using the probe, and it can be usefully utilized for early diagnosis of diseases with known biomarkers.
Hereafter, the invention will be described in more detail through examples and comparative examples. However, the following examples are to merely illustrate the present invention, and the scope of the invention is not limited by them in any ways.
1-1: Fusion Protein of Protein G and Ferritin Protein
In order to connect the genes for the ferritin protein and Protein G purchased from Promega Co., PCR was carried out using a total of three pairs of primers consisting of five primers. The primers were all purchased from Cosmogenetech (Seoul, Korea). Also, restriction enzymes Nde I, BamH I, and Xho I were purchased from New England Biolabs (Ipswich, Mass., USA).
Ferritin and Protein G were connected using a linker of 54 bp, and the linker was divided into two to carry out PCR. First, the ferritin portion was connected to Nde I and linker 1 using primer {circle around (1)} (SEQ ID NO:1) and primer {circle around (2)} (SEQ ID NO:2).
Next, primer {circle around (1)} (SEQ ID NO:1) and primer {circle around (3)} (SEQ ID NO:3) were used to connect the PCR product (Nde I+Ferritin+Linker 1) and linker 2+BamH I,
The Protein G portion was connected to BamH I and Xho I, using primer {circle around (4)} (SEQ ID NO:4) and primer {circle around (5)} (SEQ ID NO:5), and PCR conditions were the same as above. PCR was carried out under the following conditions, and each PCR product was identified by performing Agarose gel electrophoresis of the PCR products.
The terminal portions of the genes of the ferritin portion and the Protein G portion produced through PCR were each cleaved using restriction enzyme BamH I. The two genes cleaved with sticky ends were connected using a ligation enzyme as shown in
1-2: Coupling of Targeting Antibody
20 μl of 0.1 μM ferritin solution containing Protein G and fused ferritin prepared in 1) above and 3 μl of 0.1 mg/ml CLDN4 (claudin-4) anti-human antibody were mixed in a reaction vessel and then reacted at a room temperature for one hour to bond Protein G of the ferritin subunit and the antibody, thereby obtaining a probe capable of targeting a biomarker. The CLDN4 anti-human antibody was purchased from Invitrogen.
1-3: Coupling of Labeling Agent
100 μl of 0.2 μM quantum dots were added to the reaction solution obtained by the coupling of the ferritin fusion protein and the targeting antibody to mix them in the molar ratio of 1:10 and then reacted at a room temperature for one hour to produce an imaging probe for super sensitivity quantity analysis where one antibody was coupled to a number of quantum dots as shown in
Biotin was added to the C-terminal of the fusion protein of Protein G and the ferritin protein prepared in Example 1-1 using PCR. The fusion protein of Example 1-1, forward primer, and backward primer were added together as shown in Table 3, and PCR conditions are as shown in Table 4.
Next, Streptavidin was exposed at the surface of quantum dots and coupled to Biotin on ferritin. The process of exposing Streptavidin at the surface of the quantum dots is as follows. First, the surface of the quantum dots was coated with a lipid containing an SH group. 50 to 95 mole % of MHPC, 5 to 35 mole % of DPPE-PEG2000, and 1 to 15 mole % of 1,2-dipalmitoyl-sn-glycero-3-succinate containing an SH group were mixed to confer the SH group on the surface of the quantum dots. After that, the addition of SMCC rendered the SH group of the surface of the quantum dots and the maleimide group of an end portion of SMCC to be bonded by S—S disulfide bond. Lastly, the addition of Streptavidin rendered an amine group located at the other terminal of SMCC and a carboxyl group at the C-terminal of Streptavidin to be coupled, thereby exposing Streptavidin at the surface of the quantum dots.
The thus prepared fusion protein to the C-terminal of which Biotin was added and quantum dots at the surface of which streptavidin was exposed were coupled to each other by Biotin-Streptavidin reaction.
“CLDN-4” biomarker was targeted, using the target-specific probe prepared using CLDN-4 antibody as a targeting agent. CLDN-4 has been known as a typical biomarker of pancreatic cancer.
In particular, the probe was reacted to Capan-1 pancreatic cells which were fixed with 10% formaldehyde, at a room temperature. After one hour, the surface of the cells was washed repeatedly three times with PBS solution to eliminate uncoupled probes. The thus prepared image was observed using a fluorescence microscopy.
Fluorescence intensity values according to the concentrations of the quantum dots at concentrations of 1, 2, 3, 4, 5, 10, 15, 20, and 30 pmol/ml were measured using a fluorescence microscopy and as a result, the fluorescence intensity values were each 1335.2, 2670.4, 4005.6, 5340.8, 6676, 13352, 20028, 26704, and 40056, and a standard curve based on this was prepared as shown in
The amounts of biomarkers were able to be quantitatively analyzed using the standard curve. As a result of the comparison of fluorescence intensity values, the value of fluorescence intensity was 13,550 when the imaging probe for super sensitivity quantity analysis was used in
Regardless of the kinds of biomarkers, they could be quantitatively analyzed with accuracy by using a system for measuring the fluorescence intensity of quantum dots after a biomarker was targeted using ferritin and quantum dots developed in this invention, and an imaging kit was manufactured using this.
Specific experiment methods are substantially the same as example 3 and particularly, the imaging kit was used as follows: 20 μl of ferritin solution A was added to reaction vessel C, to which 3 μl of 0.1 mg/ml CLDN4 (claudin-4) anti-human antibody solution was then added as an antibody of a biomarker to be detected and reacted at a room temperature for one hour. Next, 100 μl of quantum dot solution for quantity analysis B was added and reacted at a room temperature for one hour. After targeting the reaction solution at the surface of the desired cells/tissues, fluorescence intensity was measured and compared with the standard curve of
Number | Date | Country | Kind |
---|---|---|---|
10-2013-0038147 | Apr 2013 | KR | national |