Patent Application
20220364162
The present invention relates to a single nanoparticle biosensor platform including a metal nanobridge structure and a method for constructing the same. More specifically, the present invention relates to a single nanoparticle biosensor platform including metal nanoparticles, each of which consists of two metal nanoseeds and a biomolecule anchored between the metal nanoseeds, a biosensor including the single nanoparticle biosensor platform, a method for detecting mutations using the biosensor, and a method for constructing the single nanoparticle biosensor platform including forming metal nanoparticles, each of which consists of two metal nanoseeds and a biomolecule anchored between the metal nanoseeds.
Many diseases have a genetic component; their detections therefore require a clear understanding of metabolic disorders caused by mutations for biomedical diagnostics. Most methods for diagnosing gene mutations rely on sequencing, but an optimal method for acquiring mutation information can determine the presence and identity of mutant bases without prior knowledge of the sequence, ideally without artifacts from labels and the in vitro environment. An effort has been made to find such a method with a system opposite to postreplicative mismatch repair (MMR) in many organisms, where the mismatch repair (MMR) initiation protein MutS recognizes mutations in a sequence non-specific manner depending on the introduction of other enzymes that repair MutL and mutant DNA, by a nanoplasmonic biosensor (Ma, X., Truong, P L, Anh, N. H. & Sim, S. J. Biosensors & Bioelectronics 67, 59-65 (2015)).
The reliability of nanobiosensing is generally determined by two main factors: nanomaterials for single generation and biomolecules for target recognition that are directly associated with detection sensitivity and selectivity for specific physical conditions. Among these nanomaterials, plasmonic nanoparticles have attracted interest due to their ability to interact with incident light and produce localized surface plasmon resonance (LSPR). The collective oscillation of electrons in the nanostructure at a given resonant frequency transduces changes in the local refractive index (RI) into shifts in the plasmonic bands of their absorption and scattering spectra. The sensing scale can be reduced to a single nanoparticle, contributing to single-nanoparticle sensing (sNPS); such single NP sensing (sNPS) can relay local biological information on a nanometer scale in which the limit of detection (LOD) reaches countable numbers of molecules using a very small sensing volume. In contrast, most other sensing techniques using bulk solutions or planar surfaces show a limited ability to localize and separate sensing elements and are limited by slow molecular diffusion, stochastic binding, and frequent dissociation of complexed biomolecules with consequent disequilibrium of reactions, resulting in signal fluctuations with a low signal-to-noise ratio (S/N). An sNPS sensor is a tiny probe capable of high-throughput and parallel readout in which the structure and localized sensing volume/area of the NP are essential for RI sensitivity. Systematic studies on the RI sensitivity of gold nanoparticles (NPs) with different shapes have shown that rod-like NPs exhibit the highest sensitivity to changes in RI (Truong, P. L., Ma, X. & Sim, S. J. Nanoscale 6, 2307-2315 (2014)). It has recently been demonstrated that, apart from the particle shapes, nanogap and nanobridge structures are associated with and generate strong optical signals by plasmonic coupling, further enhancing the local field to generate distinct spectral responses (Lim, D. K., et al. Nature Nanotech. 6, 452-460 (2011); Nam, J. M., Oh, J. W., Lee, H. & Suh, Y. D. Acc. Chem. Res. 49, 2746-2755 (2016)). However, synthesizing colloidal plasmonic NPs with a predefined structure is challenging due to the difficulty in manipulating atoms that are transient in solution. Moreover, chemically synthesized NPs are restricted to a highly symmetric shape with identical surface facets (e.g., nanospheres, nanorods, nanocubes, nanodisks, and others). The structural programmability of NPs could provide a powerful means to overcome sNPS limitations in sensitivity and reproducibility. Two research groups recently achieved breakthroughs in synthesis-by-design at sub-5 nm precision using a programmable biomolecule, i.e., DNA, to create well-defined nanoplasmonic particles either by casting in DNA molds (Sun, W., et al. Science 346, 1258361 (2014)) or using DNA frameworks (Ma, X., et al. Nat Commun 7, (2016)).
sNPS provides a variety of applications that make use of most of its intrinsic small sensing volume comparable to the size of biomolecules such as nucleic acids or proteins. For example, the MutS protein is 125×90×55 Å; thus, adsorption of MutS onto a single NP can drastically alter the collective oscillatory behavior of its surface electrons, resulting in wavelength shifts in the NP spectra. Since single point mutations are not substantially recognized based on PCR and other DNA chip-based assays, the specificity of the biological interaction of MutS including mismatched DNA has promoted studies on nucleotide polymorphisms. The most extensive studies are based on single-molecule fluorescence resonance energy transfer (smFRET); however, these require labor-intensive steps such as labeling of MutS or fabrication of radioactive probes for DNA. Recent studies using single-molecule imaging by atomic force microscopy are very complicated and almost impossible to apply to biomedical diagnostics. On the other hand, bulk measurements of single point mutations by gel mobility shift assay, filter/chip binding assay, nuclease protection assay, surface plasmon resonance (SPR), electrochemical assay, and quartz crystal microbalance (QCM) do not output real-time information on molecular interactions and are inefficient and time consuming.
Thus, the inventors of the present invention have earnestly and intensively conducted research to solve the problems of the prior art. As a result, the inventors have succeeded in preparing Au-bridged nanoparticles with high RI sensitivity by modifying both ends of each DNA molecule so as to bind to gold nanoseeds and crystallizing the gold in two opposite directions and have found that a single nanoparticle biosensor platform including the Au-bridged nanoparticles can be used to detect targets with high sensitivity and reliability and directly identify various mutations depending on the relative activity of MutS for the mutations. The present invention has been accomplished based on this finding.
One object of the present invention is to provide a single nanoparticle biosensor platform including a metal nanobridge structure and a biosensor including the single nanoparticle biosensor platform.
A further object of the present invention is to provide a method for detecting mutations using the biosensor.
Another object of the present invention is to provide a method for constructing the single nanoparticle biosensor platform including forming metal nanoparticles, each of which consists of two metal nanoseeds and a biomolecule anchored between the metal nanoseeds, and creating a metal nanobridge structure using the metal nanoparticles.
The present invention provides a single nanoparticle biosensor platform including a metal nanobridge structure and a biosensor including the single nanoparticle biosensor platform.
The present invention also provides a method for detecting mutations using the biosensor.
The present invention also provides a method for constructing the single nanoparticle biosensor platform including forming metal nanoparticles, each of which consists of two metal nanoseeds and a biomolecule anchored between the metal nanoseeds, and creating a metal nanobridge structure using the metal nanoparticles.
The single nanoparticle biosensor platform of the present invention can be used to not only detect targets with high sensitivity and reliability, but also to directly identify various mutations, enabling efficient diagnosis of mutations. Therefore, the single nanoparticle biosensor platform of the present invention can be utilized in a wide range of fields, including biomedical diagnostics.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly employed in the art.
In one aspect, the present invention is directed to a metal nanobridge structure, specifically a metal nanobridge structure including metal nanoparticles, each of which consists of two metal nanoseeds and a biomolecule anchored between the metal nanoseeds, and a single nanoparticle biosensor platform including the metal nanobridge structure.
In a further aspect, the present invention is directed to a method for constructing a single nanoparticle biosensor platform including forming metal nanoparticles, each of which consists of two metal nanoseeds and a biomolecule anchored between the metal nanoseeds, and creating a metal nanobridge structure using the metal nanoparticles.
In the present invention, the metal is preferably selected from the group consisting of gold (Au), copper (Cu), platinum (Pt), and palladium (Pd), more preferably gold (Au).
In the present invention, the metal nanoseeds are selected from the group consisting of nanospheres, nanorods, nanoprisms, and nanoplates.
In the present invention, the biomolecule is selected from the group consisting of single-stranded DNA, double-stranded DNA, DNA oligomer, RNA oligomer, plasmid DNA, polypeptide, and protein, preferably double-stranded DNA.
In the present invention, the metal nanoseeds preferably have a diameter of 25 nm or less.
In the present invention, the biomolecule preferably has a length of 30 nm or less.
The method of the present invention further includes reducing the metal ions with a reductant on the surface of the metal nanoparticles to grow the metal nanoparticles.
In the present invention, the reductant is hydroxylamine (NH2OH) but is not limited thereto.
In another aspect, the present invention is directed to a biosensor including the single nanoparticle biosensor platform.
The biosensor of the present invention includes a protein, preferably mismatch repair initiation protein (MutS). MutS refers to a protein that recognizes a mismatch in a nucleic acid molecule and can bind to the mismatch site. MutS is also intended to include wild-type proteins having amino acid sequences in which one or more amino acids are substituted, deleted, added, and/or inserted as long as they can recognize mismatches.
The biosensor of the present invention has a higher refractive index (RI) sensitivity than nanorods. The RI sensitivity is defined as the relative change in LSPR peak shift with respect to the refractive index change of a medium surrounding the particles. In the Examples section that follows, the RI sensitivity of the metal nanobridge structure according to the present invention was confirmed to be higher than that of nanorods, which is known to be higher than those of other nanostructures.
The biosensor of the present invention is used to detect mutations, particularly point mutations.
The biosensor of the present invention is used to specify the type of BRCA1 mutations in samples.
In yet another aspect, the present invention is directed to a method for detecting mutations, particularly point mutations using the biosensor.
The method of the present invention is used to identify proteins, preferably mutations, by binding assay of mismatch repair initiation protein (MutS) to mutant nucleic acid molecules.
The present invention will be more specifically explained with reference to the following examples. It will be appreciated by those skilled in the art that these examples are merely illustrative and the scope of the present invention is not construed as being limited to the examples. Thus, the true scope of the present invention should be defined by the appended claims and their equivalents.
1-1: Materials
Gold nanoseed (AuNS; 5 nm) solution (British BioCell International, Crumlin, UK), wash/storage buffers (10 mM PBS with 0.02% NaN3, 0.01% Tween 20, 0.1% BSA, pH 7.4; Catalog #: WB-100, Ocean NanoTech, San Diego, Calif., USA), dithiothreitol (DTT, Promega, Madison, Wis., USA) and restriction enzyme StyI (#R648A, Promega, Madison, Wis., USA), centrifuges (Microsep® and Nanosep®, Pall Life Sciences, Inc., Ann Arbor, Mich., USA), and 2-{2-[2-(2-{2-[2-(1-mercaptoundec-11-yloxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethylamine hydrochloride (OEG; Cos Biotech, Daejeon, Korea) were used. MutS protein derived from the thermophilic bacterium Thermus aquaticus was supplied by Nippon Gene Co. (Tokyo, Japan) and stored in 20 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol at −20° C. G-spin™ Total DNA Extraction Kit (#17046) was supplied by iNtRON Biotechnology (Gyeonggi, Korea). Restriction enzymes MboI (#R0147) and AluI (#R0137) were obtained from NEB (Hitchin, Hertfordshire, UK). Glycogen (#901393) was obtained from Roche (Indianapolis, Ind., USA). Poly(ethylene glycol) methyl ether thiol (PEG, Mn=800), hybridization buffer, hybridization wash pack containing single-stranded binding protein, and all other chemical reagents were purchased from Sigma-Aldrich (St. Louis, Mo., USA) and used without further purification. All glassware used in the experiments were cleaned in aqua regia solution and rinsed thoroughly with ultrapure water (18.2 mΩ·cm−1) before use. All oligonucleotides used were from Integrated DNA Technologies (Coralville, Iowa, USA). The sequences of the 8 DNA targets containing point mutations are described in Table 1. The corresponding homoduplex (perfectly matched) sequence is as follows:
The assigned names and information of single-stranded DNA (ssDNA) are shown in Table 2. The sequences of the DNA targets and probes are shown in Table 2.
aBIC (Breast Cancer Information Core): http://research nhgri.nih.gov/bic/. Nucleotide number according to GenBank U14680.1. http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=555931
bhttp://www.ensembl.org
cTwenty five nucleotides before and after the mutation point (the mutant nucleotide is marked in bold).
1-2: NP Modeling and Numerical Simulation
Modeling and optical simulations of nanostructures with spherical, rod, and dimeric shapes were performed and NPs were bridged using the software COMSOL. NPs were composed of Au; particle sizes were set as uniform to facilitate comparisons. Final dimensions were determined depending on products synthesized in experiments. Optical simulations were performed in the local dielectric environment where water-glycerol mixtures of varying weight ratios were prepared so that the RI of the surrounding medium ranged from 1.333 to 1.443 (
1-3: Conjugation of AuNSs with ssDNA
All 5′ thiol-modified oligonucleotides were incubated with a 1:100 ratio of OD of oligonucleotide to DTT solution for 15 min and purified two times with ethyl acetate. The disulfide bond of the 5′-thiol was cleaved into an active sulfhydryl form and immediately conjugated with the Au surface. Before conjugation with DNA in solution, AuNSs were coated with bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium (BSPP; 100 ml AuNS solution mixed with 100 mg BSPP for 10 h) to improve the tolerance of AuNSs to the highly ionic environment. The AuNS solution was then mixed with NaCl, which resulted in a color change from dark red to light violet. The solution was centrifuged for 30 min at 500×g and the precipitate was resuspended in 1 mL of 0.5 mM BSPP. The solution again changed color from dark red to light violet upon addition of 0.5 mL of methanol; the AuNSs were collected by centrifugation (30 min, 500×g) and dissolved in 1 mL of 0.5×TBE buffer. The concentration of AuNSs was increased to several μM, as measured with an ultraviolet-visible light-near-infrared spectrophotometer (UV-3600; Shimadzu, Kyoto, Japan); 1 OD of 5 nm AuNS is equal to 5.00×1013 particles per microliter according to the manufacturer's instructions. The AuNSs were incubated overnight at room temperature with ssDNA-1 in a stoichiometric ratio of 1:1 in 0.5×TBE buffer containing 50 mM NaCl. Thereafter, 60% glycerol was added to the solution to obtain a final mixture of 10% glycerol to prevent AuNS-ssDNA from spreading in the buffers during gel electrophoresis. AuNSs with different numbers of bound ssDNA separated into different bands on a 3% agarose gel in 0.5×TBE buffer at 10 V/cm for 1 h (
1-4: Synthesis-with-Direction of Nanostructures
Gold precursor (HAuCl4, 0.03%) and reductant (NH2OH.HCl, 1 mM) were separately dissolved in water and the pH of each solution was adjusted to 5 or 4 (±0.1) by gradually adding NaOH under a nitrogen environment. The seed for DNA-directed synthesis was produced by hybridization of AuNS-1ssDNA-1 with AuNS-1ssDNA-2 in the form of AuNS-dsDNA-AuNS. To increase hybridization efficiency, equal volumes of the two conjugates in 0.5×TBE were mixed and NaCl was added to increase ionic strength by 100 mM. The mixture was shaken overnight at 37° C. and the AuNS-dsDNA-AuNS was separated by gel electrophoresis with the same procedure as described above (
1-5: sNPS Platform Settings
The overall configuration of the sNPS system is shown in a of
1-6: Detection of Point Mutations
After mounting the glass slide in the sNPS platform, the chamber was rinsed by injecting 75% ethanol for 5 min followed by rinsing with wash/storage buffer for 20 min to remove contaminants and unbound Au-NPs. The positions of Au-bridged NPs were recorded after photographing the chamber. One NP was representative to one detection set and its optical properties were determined for each step of molecule binding. The chamber was filled with 100 nM probe DNA (e.g., Probe-GT) for 8 h at room temperature and rinsed with wash/storage buffer for 5 min before introducing target DNA (e.g., 4956A>G) at different concentrations in hybridization buffer. Unbound target was removed by rinsing with the hybridization wash pack before injecting MutS solution at the target concentrations. The binding of MutS with DNA proceeded in binding buffer (pH 7.5; 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 5 mM MgCl2) at a flow rate of 1 μL/min at 18° C. For typical detection, 100 nM target DNA was captured by Probe-GT in the chamber and reacted with 20 nM MutS protein for 2 min. Real-time imaging of single NPs with a CCD camera and RLS spectra were recorded and processed using WinSpec software (Roper Scientific). Control experiments under the same detection conditions were conducted to investigate MutS interactions with probes (without target binding) and DNA homoduplex (homoDNA; without a mutation). For the investigations on DNA interactions with nonspecific proteins (without MutS), human serum was introduced after the injection of target DNA with GT mutations. After spectral analysis, the chamber was rinsed with wash/storage buffer at 95° C. for 30 min to remove the proteins. The same serum solution containing 20 nM MutS was injected into the chamber after capturing the same target, and then the spectra were recorded again for further analysis (
1-7: Preparation and Detection of Samples from Cell Lines
The genomic DNA was extracted using G-spin™ Total DNA Extraction Kit and treated with 200 ng/ml proteinase K and 10 ng/ml RNase A at 55° C. for 30 min before purification and further restriction digestion. The digestion was performed with restriction enzymes MboI, AluI, and StyI to generate 50-60 bp nucleotides. In detail, digestion by MboI and AluI yielded fragments of 100-500 bp. Since there are StyI sites in BRCA1, the fragments were further digested by StyI to the target sample of ˜50 bp in length. The specific sites of the enzymes and the computed fragmentation maps can be found in
1-8: Demonstration of Field of Interest (FOI) of an Individual NP
The plasmonic FOI of an individual nanoparticle is defined as the effective space of plasmonic sensitivity to refractive index changes, where Equation 1 (see Section 1-10: Data analysis, infra) is applicable to calculate the molecular concentration in direct proportion to the amplitude of red shifts in λmax. The FOI was supposed to be cuboid (a of
1-9: Estimation of Average Loading Number of Probes (N*) Per NP
The N* was quantitatively predicted based on the modeling of the nanoparticle and DNA footprint (
The footprint area on the spheres (Ssphere) is indexed to be 6 nm2 according to the diameter of the sphere. The area of the two spheres (Asphere) was calculated by Asphere=A′sphere−Acontact, where A′sphere is the area of two separated spheres and Acontact is the contact area between the spheres and the cylinder; consequently, Asphere=2×4π(Dsphere/2)2−2×π(Dbridge/2)2=1178 nm2, and thus the number of probes that can be packed on the spheres was N*sphere=Asphere/Ssphere=196.
The footprint area on the outer wall of the cylinder was calculated by the equation: N*cylinder=n*short-axis×n*long-axis, where n*short-axis is the number of footprints around the circumference and n*long-axis is the number down the axis of the cylinder. However, the length of the bridge (Lbridge=2.39 nm) did not allow more than one row of probe loading along the axis of the cylinder because two rows on a non-curved surface would have a footprint spacing distance (4.72 nm; Hill, H. D. et al., ACS Nano, 3, 418-424, 2009) longer than 2.39 nm. Therefore, N*cylinder=n*short-axis×1=πDbridge/lshort-axis, where Dbridge is the circumference length and lshort-axis is the short axial length of the footprint given by lshort-axis=2×√[(3.3618 ln(Dbridge/2)+0.1616)/π]. The N*cylinder was determined to be 11, and finally, N*=N*sphere+N*cylinder=207.
Due to the immobilization of the particle on a planar substrate, it was hypothesized that only the surface above the line of “edge of effective loading” can effectively bind with DNA, which covers 59.4% of the total surface area of the particle (
1-10: Data Analysis
Changes in RI corresponding to each molecular binding step on the NP surface are expressed as LSPR λmax shifts (Δλmax):
Δλmax=m(Δn)[1−exp(−2d/Ld)] (1)
where m is the refractive index sensitivity, Δn is the change in refractive index induced by the adsorbate, d is the dielectric thickness and Ld is the electromagnetic field decay length (approximated as an exponential decay; Haes, A. J. et al., J Am Chem Soc, 124, 10596-10604, 2002). The m, Ld, and d are variables of the sNPS system for the same nanoparticles and the same lengths of probes and proteins; and therefore, Δλmax is in direct proportion to Δn, which is proportional to the concentration of the bound analytes (Starov, V. M., Nanoscience: Colloidal and Interfacial Aspects, CRC Press, Boca Raton, Fla., 2010). Based on the measurements of Δλmax, the changes in concentrations of the analytes were calculated.
The lowest concentration of MutS protein yielding a reliable Δλmax was determined as the limit of quantification (LOQ) of the sNPS procedure as follows:
LOQ=10σ/S (2)
where σ is the standard deviation of the signal and S is the slope of the calibration curve. The value of a was estimated from the standard deviation of the y intercept of the regression line.
The limit of detection (LOD) of the sNPS system for DNA target was determined as follows:
LOD=3.3σ/S (3)
The signal-to-noise ratio (S/N) was defined as the ratio of the mean (μ) to the standard deviation of Δλmax. An S/N of 5 is the threshold value to distinguish signals at 100% certainty (Bushberg, J. T. et al., Lippincott Williams & Wilkins, Philadelphia, Pa., 2012).
S/N=μ/σ (4)
In the protein-nucleic acid binding reaction, MutS binds DNA, forming the MSDNA complex. Association is a second-order reaction, involving two reactants.
MutS+DNAMSDNA (5)
Conceptually, both the binding and dissociation reactions involve straight binding. At the level of a single DNA strand, MutS association and dissociation are stochastic processes. By simple approximation, all DNA strands on the Au-bridged NP are equally available for binding. The lengths of DNA strands used indicate binding in a 1:1 stoichiometry with MutS; the time course of binding is described by a single exponential process. At the steady state, the rate of binding is equal to the rate of release:
k
binding[MutS][DNA]=kdissociation[MSDNA] (6)
where [MutS] and [DNA] are the free molar concentrations of MutS and DNA, respectively; and kbinding and kdissociation are the association and dissociation rate constants, respectively.
Before reaching the steady state, the rate of change in the concentration of the MSDNA complex is equal to the difference between its formation and dissociation rates:
d[MSDNA]/dt=kbinding[MutS][DNA]−kdissociation[MSDNA] (7)
The binding starts at the maximum rate because reactants were not consumed and then slows as reactants are consumed. The extent of the reaction over time can be expressed as follows:
[MSDNA]=[MSDNAmax]−[MSDNAmax]e−(k
The initial concentration of MSDNA ([MSDNAm]) was zero and hence the above equation can be transformed into the following:
[MSDNA]=[MSDNAmax](1−e−k
where kreaction=kbinding[MutS]+kdissociation is the observed reaction rate constant. The ratio of kdissociation (measures how fast MutS dissociates from DNA) and kbinding (measures how fast MutS binds to DNA) yields the equilibrium constant (KD, in nM) of MutS protein, which was used to evaluate the strength of bimolecular interactions and is calculated with the following equation:
Further transformation of the Equations (9) and (10) can get the equation:
where kdissociation is independent of concentration and indicates the probability that the complex will spontaneously fall apart in a unit of time (Pollard, T. D. et al., Mol Biol Cell, 24, 1103-1110, 2013).
Based on time courses of the λmax change, the time for bindings to reach half of the maximum Δλmax was evaluated by the half-time of the reaction (τ1/2):
τ1/2=ln2/kreaction (12)
2-1: Nanoparticle Design with Numerical Simulations
Since each NP functions as a signal transducer in the sNPS platform, NP structure and shape should be homogeneous and controllable. This excludes irregularly shaped nanocrystals (e.g., branched nanostars), since their formation is empirical rather than scientific based on the principles of synthesis. Furthermore, the controllability of polyhedral nanostructures is limited by the lack of chemicals that can specifically tune targeted crystal facets and thus produce NPs with a relatively high yield. Therefore, nanostructures in the shape of spheres and rods were selected as substrates for sNPS, since both can be synthesized in a uniform and scalable manner Structures consisting of nanobridges that induce distinct spectral responses and influence the magnitude of plasmonic coupling, polarization direction, signal intensity, and RI sensitivity were also introduced to explore higher RI sensitivity (a of
2-2: Synthesis-by-Design of NPs
The feasibility of “direction-specific” synthesis of gold NPs using double-stranded DNA (dsDNA) was explored by which one dsDNA (˜30 nm in length) anchored between two Au nanoseeds (AuNSs; ˜5 nm in diameter) served as a direction-specific guide for the crystallization of gold atoms (
[a]Dimensional deviation is the ratio of the standard deviation to the average size.
[b]Nanoparticles were obtained from 8 batches of synthesis and 194 particles in the plane of TEM images were analyzed.
The yield of the desired morphology was 87%, and the nanostructures were in a relatively high monodispersity (
Crystallization occurred in specific directions from the AuNS-dsDNA boundaries to the mid-point of the dsDNA strand, with nanoscale controllability defined by the length of dsDNA. This method differs fundamentally from conventional approaches involving metallizing DNA or DNA origami, in which either sequential necklaces or continuous bulges are formed with poorly controlled structural precision (>100 nm). The directional effect of DNA in the synthesis of Au-bridged NPs was evaluated by X-ray diffraction and high-resolution transmission electron microscopy (HR-TEM) (d and e of
2-3: sNPS with Au-Bridged NPs
Resonant RLS responses of a single Au-bridged NP by sNPS with a white light source were investigated (
The light generated LSPR with NPs that sufficiently enhanced light scattering to allow for direct observation of individual NPs; on the other hand, the white light illumination avoided high energy and heat that could denature target biomolecules or block molecular interactions in the microfluidic reaction chamber (a of
2-4: Sensitivity of Sensing
The sensitivity of the sNPS sensing method was investigated according to two parameters: the lowest concentration (LOD) of MutS protein enabling an LSPR λmax shift (Δλmax) to be effective within a certain detection time; and the detection time required to reach the LOD. After the MutS solution had arrived at the DNA-modified Au-bridged NPs in the microfluidic chamber, the reaction was allowed to continue for 1 min before obtaining RLS spectra for 10 s. An excess of DNA target was added to ensure complete hybridization with the probes. The effective concentration of MutS protein for the LSPR readout was 6.17 nM, corresponding to a 3.40 nm red shift in λmax in the linear range of 10-25 nM MutS (e of
2-5: Identification of Single Point Mutations
A design was made to identify eight different point mutations in BRCA1 BRCA1 gene mutations include the most important genetic susceptibility of breast cancer, the most frequent cancer of women in the world. Approximately 12% of women will develop breast cancer during their lives, with the highest risk conferred by BRCA1 mutations (59-87%). Except for few common mutations, the spectrum of BRCA1 mutations is heterogeneous in diverse populations. Eight polymorphisms of the BRCA1 gene were selected, including single-nucleotide substitutions (GT, GG, AC, TC, AA, and GA), an insertion (+C), and a deletion (−C) that are most common worldwide. The DNA sequences, mutant names, genomic locations, functional consequences, and target populations are summarized in Table 1. It was speculated that sequence-specific binding of MutS to point mutations alter distinct LSPR signals. In addition, the relative activity of MutS towards different nucleotide variants was examined Upon injection of the sNPS platform into the sensing chamber, MutS was allowed to bind to DNA-conjugated Au-bridged NPs for 150 s, and the changes in the optical response of a single NP were monitored every 1 s (a of
The relative activity of MutS to mutant DNA (Ract) was defined as the efficiency with which MutS binds to mutant DNA, expressed as Ract=K×kreaction, where K is an occupancy constant and kreaction is the rate constant of the protein-DNA interaction. This is a simple approximation of a stochastic binding event in which DNA on the Au-bridged NP is equally available for MutS; therefore, the same detection conditions allow the same K. Accordingly, Ract can be evaluated according to kreaction. The DNA probe length used (51 bp) implied 1:1 binding stoichiometry with MutS; thus, the time course of binding and disassociation can be described as a single exponential process. By fitting to the exponential equation, the kreaction (10−2 s−1) values of MutS binding to different DNA targets were 9.95±0.420, 6.15±0.208, 5.80±0.189, 4.92±0.214, 3.82±0.212, 3.60±0.243, 3.25±0.184, and 2.82±0.197 for the point mutations GT, GG, +C, AA, TC, −C, AC, and GA, respectively. By replotting the kreaction values as a function of each target DNA, the order of relative activity of MutS towards the mutations was determined as GT>GG>+C>AA>TC>−C>AC>GA (b of
2-6: Reliability of Sensing.
The crystal structure and interactions of MutS binding to a GT mismatch have been most clearly demonstrated (Groothuizen, F. S., et al. Elife 4, (2015)). Therefore, the reliability of the sNPS platform was evaluated based on a further analysis of the kreaction of MutS and GT-mutant DNA interaction to form a complex named “MSDNA”. The reaction is a second-order reaction, involving two reactants: MutS+DNA⇔MSDNA. The kinetics of MutS binding to and dissociation from DNA can be described as kreaction=kbinding [MutS]+kdissociation, where kbinding and kdissociation are the binding and dissociation rate constants, respectively, and [MutS] represents the free molar concentration of MutS (a of
2-7: Atlas of MutS Affinities to Point Mutations for Clinical Diagnosis
An atlas of protein binding affinities to DNA with four types of point mutation was further established (
As a proof-of-principle demonstration of clinical applications of the atlas, biological DNA samples were prepared from the human breast cancer cell lines, HCC1937 and MCF7, as an analyte and a control (Elstrodt, F. et al., Cancer Res, 66, 41-45, 2006), respectively, and the presence and type of a potential point mutation among the eight mutations shown in the atlas were detected (
Finally, this sNPS system was applied to detect potential point mutations in a user-assigned genomic region. A potential BRCA1 point mutation located at 43047665 on region 2 band 1 of the long arm of chromosome 17 was assigned to diagnose an ovarian cancer cell line, SNU251. The chip was fabricated with the same Au-bridged NP but with a new 64-bp probe. Interestingly, continuous shifts of the spectral peaks were observed, validating the effectiveness of the sNPS with the new probe to monitor a specific interval in the gene (
Although the particulars of the present disclosure have been described in detail, it will be obvious to those skilled in the art that such particulars are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.
The single nanoparticle biosensor platform of the present invention can be used to not only detect targets with high sensitivity and reliability, but also to directly identify various mutations, enabling efficient diagnosis of mutations. Therefore, the single nanoparticle biosensor platform of the present invention can be utilized in a wide range of fields, including biomedical diagnostics.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0095140 | Aug 2018 | KR | national |
| 10-2019-0099454 | Aug 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/010370 | 8/14/2019 | WO |
