KR102076836B1 - Composition for detecting microcystin-LR comprising magnetic beads, quantum dots and DNA intercalating dyes - Google Patents

Abstract

The present invention is a magnetic bead (magnetic bead); Two or more quantum dots (QD ₅₂₅ ), having a maximum emission wavelength at 525 nm; DNA aptamers that specifically bind to microcystin-LR; And a composition for detecting microcystine-LR comprising a PoPo3 dye, a microcystine-LR detection kit comprising the same, and a method for detecting microcystine-LR using the same.

Description

Composition for detecting microcystin-LR comprising magnetic beads, quantum dots and DNA intercalating dyes}

The present invention is a magnetic bead (magnetic bead); Two or more quantum dots (QD ₅₂₅ ), having a maximum emission wavelength at 525 nm; DNA aptamers that specifically bind to microcystin-LR; And a composition for detecting microcystine-LR comprising a PoPo3 dye, a microcystine-LR detection kit comprising the same, and a method for detecting microcystine-LR using the same.

Cyanobacterial harmful algal blooms (CyanoHABs or CHABs) are a global theme that threaten the aquatic ecosystem, water sustainability, and public health. The resulting socioeconomic losses in the United States are estimated to range from $ 2.2 to $ 4.6 billion annually. Drinking water crisis in Toledo, Ohio and Wuxi, China is a prime example. In the summer of 2014, more than 400,000 people near Lake Erie suffered from drinking water shortages for three days because of toxic cyanobacterial blooms. In Taihu Lake, China, drinking water accidents have been repeated repeatedly since the 1980s, resulting in at least a week's ban on tap water. In both cases residents were given "do not drink" warnings caused by excessive microcystin-LR (MC-LR).

MC-LR is one of the most toxic and widespread hepatoxins produced primarily by cytobacteria. For example, the lethal dose 50 (or LD ₅₀ ) of MC-LR for mice is 50 μg / kg. Microcystine is produced intracellularly and released into the surrounding water upon cell death and lysis. After release to water, toxins are found in aquatic organisms through bioaccumulation and can be delivered to humans through the food web. As a result, microcystin poisoning in the human body can occur during toxic cyanobacterial hydration. Since microcystine is primarily targeted to the human liver, acute exposure can cause serious liver damage such as liver hemorrhage and liver failure.

More than 100 microcystine variants have been reported to date, and these variants have occurred due to differences in pairs of variable L-amino acid regions. They have been identified by various chromatographic techniques (eg HPLC, LC / MS, and GC / MS) and enzyme-linked immuno-sorbent assay (ELISA). Such analytical methods can provide accurate and precise results for qualitative analysis of various microcystine congeners. However, these methods are vulnerable to interferences and cross reactivity of variants. Thus, these methods also require pretreatment steps such as separation, purification, dilution and concentration.

This time consuming pretreatment may interfere with the detection of MC-LR in environmental specimens in which various interferences and isomers coexist. In order to handle environmental water samples for MC-LR detection in a portable manner, a method with a high specificity that does not require separation and purification is advantageous. Furthermore, untreated environmental water samples are known to exhibit a relatively wide range of dissolved MC-LR concentrations of 0.1 to 10 μg / L. Qualitative analysis of these broad targets requires an iterative process of concentration and dilution prior to analysis. Thus, the method of detecting a wide range (ie having high sensitivity) is advantageous for detecting MC-LR in environmental samples.

In this regard, alternative approaches, including immunosensors, enzymatic sensors, and aptasensors, are specific to MC-LR that ensure rapidness and portability by avoiding various preprocessing steps. Is being tried. These methods allow specific and sensitive monitoring of their cognate analytes using antibodies, enzymes and aptamers as components for recognition.

Aptamers are synthetic single stranded nucleic acids (eg, DNA or RNA) capable of recognizing target molecules and are being developed as an alternative to antibodies. AN6 aptamers were first reported as having affinity for MC-LR isoforms by Ng et al. (Environ. Sci. Technol., 2012, 46: 10697-10703). When an aptamer binds to a target molecule, a configuration change of the aptamer occurs. Using the binding affinity of AN6 aptamers, various aptamer sensors have been proposed combined with colorimetric, electrochemical and fluorescence detection methods for detecting MC-LR with high binding and specificity.

Fluorescence resonance energy transfer (FRET) is a distance dependent physical process through non-radioactive energy transfer from excited donor phosphors to acceptor phosphors. This can only occur when the adjacency of the donor-receptor molecule is within 10 to 100 Hz. This makes FRET an important means for accurate and sensitive measurement of intermolecular interactions, such as ligand-receptor binding and structural changes in the nucleic acid reacting to the target molecule. Furthermore, elution (wash) free detection of signals has advantages for fast and simple analysis.

Therefore, the present inventors have studied diligently to find a simple detection method that can be performed immediately in the field while having high selectivity as well as sensitivity for detecting MC-LR toxin in an eutrophic water sample collected from the environment. Efforts have been made to use a quantum dot-aptamer complex comprising a quantum dot having an absorption and emission spectrum in a specific wavelength band, a DNA aptamer specifically binding to the target material microcystine-LR, and a dye inserted into the DNA aptamer. By selecting a combination of specific quantum dots and dyes, the quantum dot-Aptasensor was selected to confirm that high sensitivity microcystine-LR detection and quantitative analysis were possible through fluorescence resonance energy transfer, and the present invention was completed.

One object of the present invention is a magnetic bead (magnetic bead); Two or more quantum dots (QD ₅₂₅ ), having a maximum emission wavelength at 525 nm; DNA aptamers that specifically bind to microcystin-LR; And PoPo3 dyes (2-((E) -3- [1- (3-[[3- (dimethyl (3- [4-[(E) -3- (3-methyl-1,3-benzoxazol-3) -ium-2-yl) -2-propenylidene] -1 (4H) -pyridinyl] propyl) ammonio) propyl] (dimethyl) ammonio] propyl) -4 (1H) -pyridinylidene] -1-propenyl) -3-methyl It is to provide a composition for detecting microcystine-LR containing -1,3-benzoxazol-3-ium tetraiodide).

Another object of the present invention to provide a microcystine-LR detection kit comprising the composition for detecting microcystine-LR.

Another object of the present invention is to provide a water sample suspected of being contaminated with microcystine-LR; At least two quantum dots having a maximum emission wavelength at 525 nm, coupled to the surface of the magnetic beads; DNA aptamers that specifically bind to microcystine-LR bound on the quantum dots; And a first step of contacting with a probe comprising a PoPo3 dye inserted into the DNA aptamer; And a second step of irradiating light with a wavelength of 500 to 550 nm and measuring an emission spectrum of the PoPo3 dye.

In order to achieve the above object, a first aspect of the present invention is a magnetic bead (magnetic bead); Two or more quantum dots (QD ₅₂₅ ), having a maximum emission wavelength at 525 nm; DNA aptamers that specifically bind to microcystin-LR; And PoPo3 dyes (2-((E) -3- [1- (3-[[3- (dimethyl (3- [4-[(E) -3- (3-methyl-1,3-benzoxazol-3) -ium-2-yl) -2-propenylidene] -1 (4H) -pyridinyl] propyl) ammonio) propyl] (dimethyl) ammonio] propyl) -4 (1H) -pyridinylidene] -1-propenyl) -3-methyl Provided is a composition for detecting microcystine-LR comprising -1,3-benzoxazol-3-ium tetraiodide).

As used herein, the term "microcystin" refers to some freshwater cyanobacteria, mainly Microcystis. aeruginosa ) is a type of toxin produced by. To date over 100 different microcystines have been found, the most famous being microcystine-LR. Chemically, it is a cyclic heptapeptide produced through nonribosomal peptide synthase. These microcystines can be produced in large quantities during algal blooms and pose major threats to the environment as well as drinking and irrigation water raids. Specifically, microcystine is a hepatotoxic substance that can cause serious damage to the liver. Once ingested, most are transported and accumulated in the liver through the bile acid transport system, and some can remain in the bloodstream and contaminate tissue. Although it has been reported to be associated with carcinogenesis, it is not easy to demonstrate that it is difficult to accurately measure exposure to microcystine.

The target substance to be detected in the present invention, "microcystin-LR (microcystin-LR)" is a toxin produced by cyanobacteria, the most toxic form of the more than 100 toxic microcysteine variants It is widely studied in various fields such as chemistry, pharmacology, biology and environmental studies. Chemically, microcystine-LR is a non-proteinogenic amino acid of Adda, D-γ-Glu, NMe-ΔAla, D-Ala, L-Leu (L), D-β-Me-isoAsp and A compound having a cyclic structure in which seven units of L-Arg (R) are connected in sequence and L-Arg is connected to Adda, wherein another amino acid residue is substituted at the position of L-Leu and / or L-Arg. Various analogs exist. Microcystines known to date include microcystine-LR, microcystine-YR, microcystine-LY, microcystine-LW, microcystine-RR, microcystine-LF and microcystine-LA, in addition to cyano Toxins having a similar chemical structure produced from bacteria include nodularin (see Table 2). As mentioned above, microcystine-LR exhibits the highest toxicity among the toxins produced from these cyanobacteria, so it is important to selectively confirm the presence of microcystine-LR in water samples and to quantify it with high sensitivity.

The present invention selectively detects microcystine-LR, the most toxic substance among toxins caused by cyanobacterial hydration occurring in eutrophicated fresh water, from mixtures with analogues thereof, It is designed to discover a highly sensitive detection means and / or method capable of detecting microcystine-LR of. Therefore, a fluorescence resonance energy transfer (FRET) phenomenon having high detection sensitivity is used, but an object to be detected by using a quantum dot as a donor rather than an organic molecule that can be quenched over time or environment. A DNA aptamer specifically binding to the substance microcystine-LR such as AN6 (SEQ ID NO: 1) was directly bound to the quantum dot, and then a dye inserted into the DNA was used as a fluorescent receptor. Specifically, for more efficient detection, as a pair of donor and receptor which can maximize the FRET efficiency, the QD ₅₂₅ which is the quantum dot having the maximum emission wavelength at 525 nm and the dye which is inserted into the DNA which is the receptor A combination of PoPo3 dyes, having a maximum absorption wavelength at 533 nm, was chosen.

At this time, by combining a plurality of quantum dots in the micrometer-size magnetic particles can not only generate a strong signal from a single magnetic particle, but also can be used for separation if necessary.

For example, considering that individual quantum dots have a size of about 4 nm in diameter, magnetic particles having a diameter of 2.5 μm can attach up to 1.6 × 10 ⁶ quantum dot particles to the surface of a single particle. This indicates that a significant signal improvement can be achieved by introducing the magnetic particles. However, this is just one example, and may control the size of the magnetic particles to control the number of quantum dots introduced thereto.

In a particular embodiment of the invention according to size the quantum dots ipjaeul using a diameter of 2.5 μm magnetic beads and the diameter of the size of about 4 nm, by controlling these mixing ratio of was observed a signal change, the 10 ⁷ magnetic beads 4 × 10 When prepared by reacting with ^-11 moles (about 2.4 × 10 ⁶ particles) of quantum dot particles, the highest signal was seen (FIG. 2 (d)). More specifically, considering that the amount of magnetic beads is fixed and only the amount of quantum dots used is changed, the intensity of fluorescence increases as the amount of quantum dots increases, regardless of the amount of magnetic beads. When the amount of magnetic beads is fixed and only the amount of quantum dots is changed, the fluorescence intensity increases as the amount of magnetic beads increases when the amount of quantum dots is low, that is, 2 × 10 ⁻¹¹ mol or less, but the amount of quantum dots is 4 × 10 ^{At -11} moles, the fluorescence intensity decreased rather than increasing the amount of magnetic beads from 1 × 10 ⁷ to 4 × 10 ⁷ . This indicates that when the amount of the magnetic beads used is higher than a predetermined level, the fluorescence of the quantum dots may be interfered with by the presence of the magnetic beads, and thus may be reduced. On the other hand, since the quantum dots bind to the surface of the magnetic beads, the quantum dots do not overlap and bond on the magnetic beads, thus forming a single layer, and thus, considering the relative sizes of the magnetic beads and the quantum dots used, a single magnetic bead is at the maximum. The number of quantum dots that can be accommodated can be determined such that the sum of the cross sections of the quantum dots to be combined is equal to the surface area of the magnetic beads.

Therefore, it is preferable to determine the amount of use of magnetic beads and quantum dots and the mixing ratio thereof by considering the interference by the magnetic beads described above and / or the amount of quantum dots acceptable per magnetic bead comprehensively.

DNA aptamer that specifically binds to microcystin-LR in the composition is a nucleotide sequence of SEQ ID NO: 1 (5'-GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3 ').

In the composition, the quantum dots may be bound to the surface of the magnetic beads, and the DNA aptamer may be bound on the quantum dots. An example of a composite structure in which they are bonded to each other is shown schematically in FIG. 1. FIG. 1 is a diagram illustrating a coupling relationship between these components. Each of the relative scales may be different from the actual size.

Meanwhile, the "PoPo3 dye" is 2-((E) -3- [1- (3-[[3- (dimethyl (3- [4-[(E) -3- (3-methyl-1,3) -Benzoxazol-3-ium-2-yl) -2-propenylidene] -1 (4H) -pyridinyl] propyl) ammonio) propyl] (dimethyl) ammonio] propyl) -4 (1H)- Pyridinylidene] -1-propenyl) -3-methyl-1,3-benzoxazole-3-ium tetraiodide (2-((E) -3- [1- (3-[[3 -(dimethyl (3- [4-[(E) -3- (3-methyl-1,3-benzoxazol-3-ium-2-yl) -2-propenylidene] -1 (4H) -pyridinyl] propyl) ammonio) propyl] (dimethyl) ammonio] propyl) -4 (1H) -pyridinylidene] -1-propenyl) -3-methyl-1,3-benzoxazol-3-ium tetraiodide) Has the chemical formula shown. The PoPo3 dye is intercalated in the DNA aptamer in the absence of microcystine-LR and desorbed upon contact with microcystine-LR. Specifically, when PoPo3 is inserted into the DNA aptamer, the distance from the quantum dot to which the aptamer is attached may be maintained within 10 nm, which may indicate fluorescence resonance energy transfer, while the complex is in contact with the microcystine-LR. In this case, the DNA aptamer specifically binds to the microcystine-LR and releases the PoPo3 dye inserted therein, so that the fluorescence resonance energy transfer from quantum dots to PoPo3 no longer occurs.

[Formula 1]

For example, the composition may exhibit an emission spectrum in a region consistent with the fluorescence spectrum of the PoPo3 dye when it is irradiated with light of 500 to 550 nm wavelength in the absence of microcystine-LR.

On the other hand, the composition, when in contact with the microcystine-LR, may exhibit an emission spectrum of the quantum dot QD ₅₂₅ when excited by irradiation with light of 500 to 550 nm wavelength.

For example, the composition of the present invention, in order to bind each component in the desired order, the surface of the magnetic beads and one end of the aptamer is modified with an amine group, the surface of the quantum dot QD ₅₂₅ is modified with a carboxyl group, or the surface of the magnetic beads And one end of the aptamer may be modified with a carboxyl group, and the surface of the quantum dot QD ₅₂₅ may be modified with an amine group.

As such, the magnetic bead surface, the quantum dot, and the quantum dot surface and the aptamer modified or modified with an amine group or a carboxyl group, respectively, are N-hydroxysuccinimide (NHS) through a carboxyl group and an amine group contained at each end thereof. It can be bound by reaction with ethylcarbodiimide hydrochloride (EDC).

For example, the compositions of the present invention i) aminated magnetic beads (aminated magnetic beads) and the quantum dot (carboxylic quantum dots) modified with a carboxyl group at a predetermined ratio, for example, 10 ⁷ magnetic beads of 1 to 4 × 10 ^{- After} mixing with ¹¹ moles of quantum dots and reacting with the addition of NHS and EDC to obtain an intermediate having a plurality of quantum dots bonded to the surface of the magnetic beads, ii) the intermediate in the presence of NHS and EDC micro-modified at one end with an amine group By reacting with cystine-LR specific DNA aptamers to allow DNA aptamers to bind via an amine group at one end of the unreacted carboxyl groups on the quantum dots that do not participate in binding to the magnetic beads. Aptamer complexes can be prepared. Insertion of PoPo3 dye can be achieved through a simple process of immersing the complex in a PoPo3 dye solution.

Furthermore, the present invention provides a combination of a fluorescent donor and a fluorescent receptor optimized for detecting a target substance such as microcystine-LR by FRET using a complex including magnetic beads, a quantum dot as a fluorescent donor, a DNA aptamer and a dye which is a fluorescent receptor. Suggest. To this end, in a specific embodiment of the present invention, a comparative example includes a quantum dot (QD ₅₆₅ ) as a fluorescent donor, but as a corresponding fluorescent acceptor, carboxy-X, which is an organic phosphor that exhibits fluorescence by absorbing at the maximum emission wavelength of the quantum dot. Attempted detection of target material using a complex covalently bound to aptamer with carboxy-X-rhodamnine (ROX). As a result, the detection system using the complex increased the concentration of the target material to a certain level. It was confirmed that the specific level of the target material was not detected by displaying a signal at the same level as the control group, that is, without the target material (Comparative Example 1 and FIG. 11). This indicates that even with the FRET based on the quantum dot-aptamer-dye complex, the detection of the target substance may not be possible depending on the combination of the fluorescent donor and the receptor, and specifically, a fluorescent donor having a spectrum suitable for exhibiting the FRET phenomenon. Selecting a combination of and a receptor indicates that the detection of the target substance by FRET may not be possible. Thus, even a person skilled in the art cannot easily derive a system for detecting a target substance by FRET using a specific combination of quantum dot-aptamer-dye complexes. Based on this unpredictability, in the present invention, a combination of a quantum dot (QD ₅₂₅ ) having a maximum emission wavelength at 525 nm, a DNA aptamer specifically binding to microcystine-LR and a PoPo3 dye inserted into the DNA aptamer It is suggested that this is the optimal combination for the specific and sensitive detection of microcystine-LR.

The second aspect of the present invention provides a microcystine-LR detection kit comprising the microcystine-LR detection composition of the first aspect.

As described above, the composition for detecting microcystine-LR of the present invention comprises: magnetic beads; Two or more quantum dots (QD ₅₂₅ ), having a maximum emission wavelength at 525 nm; DNA aptamers that specifically bind to microcystin-LR; And a PoPo3 dye, wherein the quantum dots are bound to the surface of the magnetic beads, and the DNA aptamer may be provided in the form of a complex bound on the quantum dots. Alternatively, the kit of the present invention includes the composition in the form of a precursor, that is, in the form of a precursor of magnetic beads modified at an amine group, an aptamer modified at an amine group at one end, and a quantum dot QD ₅₂₅ modified at a carboxyl surface, It can be provided with N-hydroxysuccinimide (NHS) and ethylcarbodiimide hydrochloride (EDC) to induce binding between these components. PoPo3 dyes may also be provided embedded in the complex, or may be provided separately in the form of a solution or powder, optionally with a solvent.

A third aspect of the invention provides a sample of water suspected of being contaminated with microcystine-LR; At least two quantum dots having a maximum emission wavelength at 525 nm, coupled to the surface of the magnetic beads; DNA aptamers that specifically bind to microcystine-LR bound on the quantum dots; And a first step of contacting with a probe comprising a PoPo3 dye inserted into the DNA aptamer; And a second step of irradiating light with a wavelength of 500 to 550 nm and measuring an emission spectrum of the PoPo3 dye.

For example, the detection method of the present invention comprises a magnetic bead prior to the first step; At least two quantum dots having a maximum emission wavelength at 525 nm, coupled to the surface of the magnetic beads; DNA aptamers that specifically bind to microcystine-LR bound on the quantum dots; And irradiating light having a wavelength of 500 to 550 nm to the probe including the PoPo3 dye inserted into the DNA aptamer and measuring the emission spectrum of the PoPo3 dye. The spectrum measured through step 1-1 may be used as a reference for determining whether and / or content of microcystine-LR in the sample from the spectrum measured thereafter.

Specifically, when the emission signal of the spectrum measured from step 2 is lower by comparing the spectrum measured from step 2 with the spectrum measured from step 1-1, it is determined that the corresponding water sample contains microcystine-LR. The third step may be further included.

Furthermore, by calculating the concentration of microcystine-LR contained in the water sample from the reduced degree of the luminescence signal of the spectrum measured from the second step compared with the spectrum measured from the step 1-1, the micro in the water sample Cystine-LR can be quantitatively analyzed.

A fourth aspect of the present invention is a light source unit capable of irradiating light of 500 to 550 nm wavelength; A sample injection unit positioned in a path of light irradiated from the light source unit; A microcystine-LR detection apparatus comprising: a detector configured to detect an optical signal having a wavelength selected from a range of 550 to 610 nm spaced apart from the sample injection unit by a predetermined distance in a direction perpendicular to the light source unit. It provides an analysis device for.

As described above, the composition for detecting microcystine-LR based on FRET using the complex of the quantum dot and the aptamer of the present invention has a very high luminous efficiency and a low detection limit, and thus can be detected without the help of a complex amplification apparatus. The analysis device may be implemented as a small portable device.

The relative position of each component mentioned in the analysis apparatus means an optical path, and a series of lenses and / or mirrors, or the like, may be introduced on the optical path to guide and collect incident light and fluorescent signals. It can be changed by the intervention of these optics. That is, the analysis apparatus of the present invention is FRET, that is, to measure the fluorescence, as long as it can induce light emitted in a direction perpendicular to the light incident on the sample to the detection unit in order to minimize the interference by the incident light, The relative position of each component is not limited to the absolute position and / or arrangement described above.

The composition for detecting microcystine-LR of the present invention has high selectivity and / or sensitivity to microcystine-LR using FRET including a combination of a quantum dot and a PoPo3 dye having a maximum emission wavelength at 525 nm as a fluorescent donor and a receptor. Since the detection limit can be detected quantitatively up to a significantly lower detection limit than the conventional ELISA assay, it can also be detected by a portable analysis device, which is useful for in situ analysis of trace amounts of microcystine-LR contained in a real environmental water sample. Can be used.

1 is a view schematically showing a method of manufacturing a FRET-based QD-apta sensor for detecting MC-LR and a detection principle using the same.
FIG. 2 shows (a) circular dichroism (CD) spectral change of aptamer by MC-LR addition, (b) CD spectral change of aptamer by PoPo3 dye addition, (c) into aptamer Optimization of PoPo3 dye concentration for efficient insertion, (d) Optimal ratio of magnetic beads (MB) and quantum dots (QD) for the preparation of MB-QD ₅₂₅ , (e) FRET configuration Feasibility test and (f) MB-QD ₅₂₅ -aptamer complex showing the stabilizing effect.
FIG. 3 shows fluorescence changes of PoPo3 dyes by insertion into aptamers.
Figure 4 is a diagram showing a microscope image of the FRET-based QD-Abtta sensor. (a) shows scanning electron microscopic images of MB-QD ₅₂₅ complex, and (b) shows confocal microscopic images of MB-QD ₅₂₅ -aptamer with PoPo3 dye. .
Figure 5 shows the results of (a) the change in the normalized fluorescence of the FRET-based QD-apta sensor during the reaction and (b) the sensitivity confirmation for MC-LR detection in the reaction of 6 hours.
Figure 6 is a diagram showing the results of the sensitivity detection of the FRET-based QD- apta sensor in the target material of 1 ng / mL concentration.
7 shows a standard curve of MC-LR quantification using an ELISA kit.
8 is a diagram showing the results of MC-LR analysis of Microcystis aeruginosa UTEX 2388 ( M. aeruginosa ) cells using FRET-based QD-Apta sensor. (a) shows the algae growth curve according to the MC-LR concentration of the culture medium in the absence of MC-LR (lime green) and QD-apta sensor (blue) and ELISA assay (red), and (b) shows the growth of M. aeruginosa . Specific growth rate, (c) shows Pearson correlation of MC-LR concentration between QD-Apta sensor and ELISA assay.
Fig. 9 shows (a) a site map showing sampling points, (b) NC1 and (c) NC2, which are samples taken from a negative control point, and (d) S1, (e) S2, which are samples taken from a research site. ) S3 and (g) The figure which showed the result of observing S4 visually.
10 is a diagram showing the results of MC-LR analysis in environmental water samples using FRET-based QD-Apta sensor and ELISA assay. (a) shows the MC-LR concentration in the sample collected at each point, and (b) shows the Pearson correlation of MC-LR concentration between QD-Apta sensor and ELISA assay.
FIG. 11 illustrates DEHP ((di-2-ethylhexyl) phthalate) detection results using a QD-apta sensor-based detection method using an organic phosphor as a receptor.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example  1: FRET based Quantum dots - Aptamers  Sensor Design and Operation Principle

The QD-Aptasensor (QD-Aptasensor) is composed of magnetic beads, quantum dot nanoparticles and aptamers. As shown in FIG. 1, carboxyl-modified carboxylic quantum dot nanoparticles (QD ₅₂₅ ) were immobilized on the surface of aminated magnetic beads (MB) (FIGS. 1A and 1B). . The resulting complex (MB-QD ₅₂₅ ) was combined with aminated aptamers having a high affinity for microcystin-LR (MC-LR) molecules (FIG. 1C). PoPo3 dye was then intercalated into the aptamer of the MB-QD ₅₂₅ -aptamer complex (FIG. 1D). QD ₅₂₅ (fluorescent donor; donor fluorophore) and PoPo3 dyes (fluorescent acceptor; acceptor fluorophore) are excited at 360 nm and 530 nm wavelengths to emit fluorescence at 530 nm and 561 nm, respectively. This allows the PoPo3 dye to be released via fluorescence resonance energy transfer (FRET) from QD ₅₂₅ excited at 360 nm. As the aptamer captures MC-LR molecules, the PoPo3 dye is a MB-QD ₅₂₅ -aptamer. As a result, the PoPo3 dye is separated from QD ₅₂₅ nanoparticles and does not release via FRET media at 360 nm.

Figure 1 shows the MC-LR quantitative analysis by FRET-based fluorescence change of the MB-QD ₅₂₅ -Aptamer-PoPo3 design. For this purpose, two phosphors, QD ₅₂₅ and PoPo3 dyes, were selected as FRET pairs. The excitation spectrum of the PoPo3 dye was superimposed with the emission spectrum of the QD ₅₂₅ nanoparticles. QD ₅₂₅ nanoparticles were used as excited molecular fluorophores (donors). The excitation energy of QD ₅₂₅ was transferred to a dimeric cyanine PoPo3 dye molecule (acceptor fluorophore), already inserted into the aptamer. As a result, PoPo3 dye was released via FRET of QD ₅₂₅ nanoparticles by intermolecular long-range dipole-dipole coupling. The binding of MC-LR induced a structural change in the aptamer, resulting in the release of PoPo3 dye molecules from the construct, leading to a decrease in PoPo3 dye release. Since FRET efficiency depends on the donor-acceptor proximity of the donor, this distance-dependent process, which is suitable for binding-induced separation of donor-receptor pairs, prevents highly sensitive detection of MC-LR. Allowed.

Example  2: FRET Based on QD-Apta Sensor  Produce

2-1. MC- LR  About Aptamer  Structural change

Single stranded DNA (ssDNA) aptamer AN6 (5'-NH ₂ -GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3 'for detection of MC-LR) , Bioneer Corporation, Daejeon, Korea). Aptamers are modified by attaching amine groups from the original form, thereby binding the aptamer's binding affinity to MC-LR with circular dichroism (CD) spectropolarimetry. Was tested. 60 μL of aptamer at 10 μmole / L concentration of Tris-HCl buffer at 0.02 mole / L concentration containing 0.01% SDS, 20 mmole / L MgCl ₂ , 40 mmole / L KCl, and 100 mmole / L NaCl ( Tris buffer, pH 8.0). Subsequently, various concentrations (10 fg / mL, 1 pg / mL, and 10 pg / mL) of MC-LR solution were added to the solution in 15 μL (total volume 300 μL) and incubated overnight at room temperature. The aptamer solution was transferred to a quartz cuvette with an optical path length of 1 mm. 25 ° C. CD spectra were scanned using a Jasco J-1500 spectrophotometer (Jasco Inc., Easton, MD, USA) equipped with a Peltier thermostatic cell holder. CD spectral scans were performed at a rate of 50 nm / min from 350 nm to 200 nm. Each spectrum was corrected for baseline (buffer only) and was obtained by accumulating three times data. The experiment was performed twice.

Degree As shown in 2a, the CD spectrum indicates that the aptamer is a typical B-form of DNA with positive and negative bands at 275 nm and 245 nm, respectively. More specifically, positive band peaks of B-type DNA appear around 260-280 nm by base stacking of DNA, while negative bands are weak due to the right-handed helicity of DNA. It appears at 245 nm. As shown by the red dotted circle in FIG. 2A, the interaction of aptamer with MC-LR induced a change in the CD spectrum at ˜245 nm. After adding MC-LR, the negative CD band at ~ 245 nm showed a decrease from -0.8916 mdeg (aptamer alone) to -0.6477 mdeg (aptamer with 10 pg / mL of MC-LR). , Positive bands at ˜275 nm were found to be less affected. In general, changes in negative CD bands can be observed in DNA insertion events. Specifically, the decrease in intensity of the negative CD band can occur when the target molecule is inserted perpendicular to the helical DNA axis. The peak at ˜245 nm is the only negative CD band in FIG. 2A and there is a decrease in intensity at that peak, so it can be concluded that MC-LR molecules are inserted perpendicular to the base pair and bind to the aptamer.

2-2. By adding PoPo3 dye Aptamer  Structural change

Conformational change of aptamer according to the addition of PoPo3 dye was confirmed by CD spectroscopic polarization as in Example 2-1. The solution was prepared in the same manner as in Example 2-1. Briefly, 10 μmole / L aptamer aliquots (60 μL) were transferred to 1.5 mL vials containing 210 μL of 0.02 mole / L Tris-HCl buffer (pH 8.0). 30 μL of iodide PoPo ^™ -3 dye (Dimeric cyanine nucleic acid stains, Invitrogen, Carlsbad, Calif., USA) was then added to the vial to a final concentration of 0.1 μmole / L. The vials were incubated overnight at room temperature. The solution was transferred to a cuvette and the CD spectrum was measured in the same manner as in Example 2-1.

2-3. Aptamers  Mine PoPo3  Insertion of dye

 Aptamers were suspended in varying amounts (0, 10, 50, 100, 200, and 500 pmoles) in 90 μL of 0.02 mole / L Tris buffer. The solution was transferred to an opaque 96-well microplate (Thermo Fisher Scientific, Wilmington, DE, USA). Subsequently, 10 μL of PoPo3 dye was added to the microplate to prepare Tris buffer solution at final concentrations of 0.1, 1, 2.5, 5, and 10 μmole / L. The fluorescence of the PoPo3 dye at 561 nm with 530 nm excitation wavelength was measured using SpectraMax M2 spectrofluorometer (Molecular Devices, Sunnyvale, Calif., USA) and recorded for 2 hours at 10 minute intervals. All experiments were performed three times.

Degree As indicated by the red dotted circle in 2b, the negative CD peak of the aptamer at ˜245 nm was changed by addition of PoPo3 dye at a concentration of 0.1 μmole / L. In this same way, the decrease in the value at the negative peak indicates that PoPo3 has been inserted into the aptamer by insertion in the vertical direction. Insertion of PoPo3 dye into the aptamer was used to determine the optimal aptamer concentration by testing with varying concentrations of PoPo3 dye for the unit usage of the aptamer (FIG. 2c). As determined in FIG. 3, after incubating the PoPo3 dye and the aptamer together for 10 minutes, the fluorescence signal (RFU _{561 nm} ) of the inserted PoPo3 dye increased proportionally with the amount of aptamer at various PoPo3 dye concentrations. Linear regression analysis calculated the relationship between the aptamer amount and the PoPo3 dye concentration. A coefficient of determination (r ² ) between the fluorescence signal (RFU _{561 nm} ) and the amount of aptamer was obtained. Both PoPo3 dyes at 2.5 and 5 μmole / L concentrations showed the highest crystal constants of 0.992 and 0.993, respectively. Although PoPo3 dyes at 10 μmole / L concentration showed the highest fluorescence, PoPo3 dyes at 5 μmole / L concentration were selected for efficient insertion at various aptamer concentrations (Fig. Arrow of 2c).

2-4. magnetism Bead  versus Quantum dots  Optimal ratio

A range of the ratio between in order to obtain the highest fluorescent signal of the _{QD 525, MB (1, 2} , and 4 × 10 ⁷ more particles, Invitrogen) and the QD ₅₂₅ (1, 2, and 4 × 10 ^-11 mol, Invitrogen) Tested. Aminated MB was combined with QD ₅₂₅ modified with carboxyl groups by reaction with NHS / EDC (N-hydroxysuccinimide / ethylcarbodiimide hydrochloride, Sigma-Aldrich, Saint-Louis, MO, USA) for 2 hours. Unbound QD ₅₂₅ nanoparticles were removed by washing with 0.02 mole / L Tris buffer. The resulting MB-QD ₅₂₅ complex was redispersed in 200 μL of the same buffer and transferred to an opaque 96-well microplate. Excited at 360 nm wavelength and fluorescence was measured at 530 nm. All experiments were performed four times.

Various mixing ratios of MB particles per unit QD ₅₂₅ were tested to optimize the formation of the MB-QD ₅₂₅ complex. Degree Regarding 2d (red dashed circle), the highest fluorescence signal (RFU _{530 nm} ) of QD ₅₂₅ was achieved at 4 × 10 ⁻¹¹ moles of QD ₅₂₅ and MB of 10 ⁷ particles. In 4 × 10 ⁻¹¹ moles of QD ₅₂₅ , fluorescence was reduced by the addition of more MB particles, from 10 ⁷ to 4 × 10 ⁷ . This indicates that the fluorescence of QD ₅₂₅ may be interfered by MB particles due to the size difference. The MB and QD ₅₂₅ particles used had diameters of ˜2.5 μm and ˜4 nm, respectively. Mixing ratios of MB (10 ⁷ particles) and QD ₅₂₅ (4 × 10 ⁻¹¹ moles) particles were selected and used in subsequent experiments. On the other hand, when the amount of QD ₅₂₅ particles used was relatively small (1 or 2 × 10 ⁻¹¹ moles), the larger the number of MB particles used, the more the fluorescence signal measured increased. It is believed that this is because it enables signal collection.

2-5. Feasibility of setting FRET

The FRET configuration (FIG. 1D) was demonstrated by the insertion of PoPo3 dyes into the MB-QD ₅₂₅ -aptamer complex. MB-QD ₅₂₅ composites were prepared according to the optimal ratio of MB (1 × 10 ⁷ particles) and QD ₅₂₅ (4 × 10 ⁻¹¹ mole). Aliquots (100 μL) of the MB-QD ₅₂₅ complex were transferred to six 1.5 mL vials. 20 μL of an aptamer solution (10 μmole / L) having an amine functional group at the 5 ′ end was added to three vials, and then MB-QD ₅₂₅ -aptamer complex was formed through NHS / EDC reaction as follows.

Unbound aptamers were removed by washing twice with 0.02 mole / L Tris buffer and the MB-QD ₅₂₅ -aptamer complex was redispersed in the same buffer. For comparison, the MB-QD ₅₂₅ complex was prepared simultaneously in three different vials as negative controls. Each solution in the vial was transferred to a microplate and PoPo3 dye (5 μmole / L as final concentration) was added to the plate. Plates were incubated for 10 minutes at room temperature with agitation at 1,200 rpm in a MixMate shaker (Eppendorf, Hamburg, Germany). By using a SpectraMax M2 spectral optical fields excited by 360 nm wavelength at 561 nm was measured FRET- mediated fluorescence emission _(561nm RFU) of PoPo3 dyes. All experiments were performed three times.

The feasibility of FRET-based PoPo3 dye release was tested and the results are shown. 2e. MB-QD ₅₂₅ -Aptamer was incubated with PoPo3 dye for insertion. After washing the unbound PoPo3 dye, the fluorescence of the measured PoPo3 dye (RFU _{561 nm} = 258 ± 6) indicated that significant insertion of the PoPo3 dye occurred in the aptamer. As expected, the aptamer-free MB-QD ₅₂₅ (voice control) showed a signal value corresponding to background noise (RFU _{561 nm} = 43 ± 18). This indicates that the fluorescence of the PoPo3 dye was successfully released by excitation by the release of QD ₅₂₅ , which was mostly derived from the insertion of PoPo3 dye into the MB-QD ₅₂₅ -aptamer. The background noise signal was less than 20%.

2-6. MB-QD ₅₂₅ - Aptamer  stabilize

Sodium borohydride (NaBH ₄ ) solution was used to stabilize the MB-QD ₅₂₅ -aptamer complex. NaBH ₄ solution was freshly prepared at a final concentration of 132 mmole / L in 2 × SSC containing 0.05% SDS. An aliquot (100 μL) of MB-QD ₅₂₅ -aptamer complex was transferred to a 1.5 mL vial. The NaBH ₄ solution (100 μL) was added to half of a total of 36 vials, and the other half of the vial was ultra high purity distilled water (ultrapure distilled water, DNase / RNase / Protease free, Intron Bio-technology, Gyeonggi, Korea). ) Was added. These vials were incubated for 5-30 minutes at room temperature with stirring at 1,200 rpm in a MixMate shaker. After stabilization for different reaction times, the MB-QD ₅₂₅ -aptamer complex was washed twice with 0.02 mole / L Tris buffer and redispersed in the same buffer. MB-QD ₅₂₅ -aptamer complex was transferred to an opaque 96-well microplate and PoPo3 dye (5 μmole / L as final concentration) was added to the solution. After incubation for 10 minutes, the fluorescence signal of QD ₅₂₅ and FRET-based PoPo3 dyes was measured at 530 nm and 561 nm after excitation at the same 360 nm wavelength using a SpectraMax M2 spectrofluorometer. Using each fluorescence, normalized fluorescence was determined by the following equation (1). All experiments were performed three times.

(1)

(One)

Degree 2f shows the stabilization result of MB-QD ₅₂₅ -Aptamer by NaBH ₄ over time. Normalized fluorescence of MB-QD ₅₂₅ -aptamer (PoPo3 / QD ₅₂₅ ) was higher (ie, improved) when the complex was stabilized by NaBH ₄ (closed circle in FIG. 2F). In particular, 5 min stabilization (red arrow in FIG. 2F) showed the highest normalized fluorescence and was therefore selected for use in subsequent experiments. The functional group of MB-QD ₅₂₅ -aptamer was inactivated by capping with NaBH ₄ , a reducing agent. This may block nonspecific binding or aggregation of PoPo3 dyes.

2-7. QD - Apta sensor  Microscopic image analysis

To visually confirm the structure of the complex, microscopic image analysis was performed, including scanning electron microscopy and confocal microscopy. Example 2 prepared MB-QD ₅₂₅ and MB-QD ₅₂₅ -aptamer complexes, respectively. The surface of the MB-QD ₅₂₅ composite was subjected to field emission-scanning electron microscopy (FE-SEM, SU-70, Hitachi High Technologies Corp., Tokyo, Japan) at an acceleration voltage of 30 kV. Analyzed. The formation of the MB-QD ₅₂₅ -aptamer complex was confirmed by confocal microscopy. For visual verification of the aptamers, PoPo3 dye was added to the MB-QD ₅₂₅ -aptamer complex. After insertion of the PoPo3 dye into the aptamer, fluorescence images of the aptamer containing QD ₅₂₅ and PoPo3 dyes were super resolution confocal microscopes (TCS SP8 STED, Leica Microsystems, Wetzlar, Germany) at 530 nm and 560 nm emission wavelengths, respectively. ) Was taken.

Formation of the QD-apta sensor complex was confirmed by microscopic analysis (FIG. 4). As indicated by the arrows in FIG. 4A, SEM images showed that QD ₅₂₅ nanoparticles were immobilized on the surface of MB particles. In addition, it was shown visually through confocal microscopy (Fig. 4b). The bottom left figure of FIG. 4B shows QD ₅₂₅ nanoparticles that only emit at 530 nm. The bottom right plot of FIG. 4B shows the embedded PoPo3 dyes taking FRET based 560 nm emission under 360 nm excitation (FIG. 1D). Confocal microscopy images showed the successful insertion of PoPo3 dyes into the MB-QD ₅₂₅ -aptamer conjugate. Aptamers were visualized by taking fluorescence images of FRET-based PoPo3 dyes.

Example  3: QD - Apta sensor  Sensitivity Check

An aliquot (160 μL) of MB-QD ₅₂₅ -aptamer complex was transferred to a microplate and PoPo3 dye (20 μL) was added to the microplate for insertion. After inserting PoPo3 dye into the aptamer for 10 minutes, 10-fold serial diluted MC-LR (10 ^-4 to 10 ² ng / mL, Enzo Life Sciences, New York, NY, USA) was added to a total volume of 200 μL. It was added to the microplate. As a negative control (NC), ultrahigh-purity distilled water was added instead of MC-LR. The microplates were incubated for 10 hours at room temperature with stirring at 1,200 rpm in MixMate. For a given incubation time, fluorescence of QD ₅₂₅ and FRET-mediated PoPo3 dyes was determined at 530 nm and 561 nm under 360 nm excitation using a SpectraMax M2 spectrofluorometer (0, 0.17, 1, 2, 4, 6, 8). , And 10 hours). Subsequently, normalized fluorescence (PoPo3 / QD ₅₂₅ ) of the negative control group ( NF _NC ) and the sample ( NF _Sample ) was calculated according to Equation (1). The relative decrease in normalized fluorescence due to the difference between NF _NC and NF _Sample (Δ (PoPo3 / QD ₅₂₅ )), as shown in Equation (2) below, is the sensitivity and selectivity of the method proposed in the present invention. (selectivity) was used for verification (FIGS. 5 and 6). All experiments were performed three times.

(2)

(2)

FRET-based QD-Apta sensors were able to detect and quantify MC-LR molecules. Relative normalized fluorescence (Δ (PoPo3 / QD ₅₂₅ )) was monitored during the 10 hour reaction and the signal increased with time (FIG. 5A). This indicates that FRET can measure time-dependent fluorescence changes. As shown in FIG. 5A, the relative normalized fluorescence increased rapidly at the beginning of the reaction and then nearly saturated after the 6 hour reaction. Therefore, relative normalized fluorescence was used at 6 hours to further demonstrate the sensitivity of the QD-Apta sensor for MC-LR detection. With reference to FIG. 5B, the relative normalized fluorescence was linearly proportional to the concentration of MC-LR in the range of 10 ⁻⁴ to 10 ² ng / mL (ppb or nmole / L). A regression of y = 0.15 log ₁₀ x +0.93 (r ² = 0.94) is shown. At this time, the relative normalized fluorescence is the y -axis and the MC-LR concentration is the x -axis. The analytical limit of detection (LOD) was 10 ⁻⁴ ng / mL (nmole / L), which is much lower than the WHO guideline level for drinking water, 1 ng / mL. Table 1 below summarizes the detection limits for various aptamer-based MC-LR quantification methods known in the art. This indicates that the FRET-based QD-Apta sensor according to the present invention has the highest level of sensitivity to MC-LR molecules compared to other aptamer-based analytical methods.

Detection method Detection limit
(ng / mL) literature Colorimetric Apta Sensor 0.37 J. Hazard. Mater. 304 (2016) 474-480 Colorimetric Apta Sensor 0.05 Biosens. Bioelectron. 68 (2015) 475-480 Electrochemical Apta Sensor 11.8 × 10 ^-3 Environ. Sci. Technol. 46 (2012) 10697-10703 Microcantilever Array Biosensor One Sensor. Actuat. B-Chem. 260 (2018) 42-47 Electrochemical Aptoxy Sensor 0.04 Sensors (Basel) 16 (2016) E1901 Volt Amp Apta Sensor 1.9 × 10 ^-3 Anal. Chem. 86 (2014) 7551-7557 Electrochemical Impedance Biosensor 1.8 × 10 ^-2 Talanta 103 (2013) 371-374 Fluorescent Apta Sensor 0.139 Talanta 166 (2017) 187-192 Upconversion Based Fluorescent Apta Sensor 2 × 10 ^-3 Biosens. Bioelectron. 90 (2017) 203-209 Dual FRET Apta Sensor 0.1 Anal. Bioanal. Chem. 407 (2015) 1303-1312 FRET-based QD-Apta Sensor 10 ^-4 The present invention

Example  4: QD - Apta sensor  Selectivity check

An aliquot (160 μL) of MB-QD ₅₂₅ -aptamer complex was transferred to a microplate and PoPo3 dye (20 μL) was added to the microplate. For selectivity confirmation, a total of seven MC-LR analogs (MC-YR, MC-LY, MC-LW, MC-RR, MC-LF, MC-LA, and Nodularin) were obtained from Enzo Life Sciences Inc. Obtained. Specific information of the analogs is listed in Table 2 below. 20 μL of each analog compound was added to the QD-AptaSensor reaction to a final concentration of 1 ng / mL. The concentration of 1 ng / mL corresponds to the microcystine concentration on the World Health Organization (WHO) guidelines. In addition, MC-LR and ultrapure distilled water were added to the reaction as a positive control and a negative control, respectively. The microplates were incubated for 6 hours at room temperature with stirring at 1,200 rpm in MixMate. After incubation, fluorescence of QD ₅₂₅ and FRET-based PoPo3 dyes was measured using SpectraMax M2 spectrofluorometer. Relative normalized fluorescence was determined according to equation (2) above. All experiments were performed three times.

The selectivity of the FRET based QD-Apta sensor was verified using MC-LR and its analogous chemicals. As shown in Table 2, seven analogs having chemical structural similarities to MC-LR (specifically, MC-YR, MC-LY, MC-LW, MC-RR, MC-LF, MC-LA, and Nodu) Larin (nodularin) was selected. As shown in FIG. 6, MC-LR was selectively detected at 1 ng / mL concentration by FRET based QD-Apta sensor. The relative normalized fluorescence of the highest MC-LR represents the highest affinity of the MC-LR compared to other analogs. When the affinity for MC-LR was set to 100%, the relative affinity for MC-YR and MC-LA was all close to 0%. On the other hand, MC-LY, MC-RR, and noduline showed a relative affinity of about 13-15%, and MC-LW and MC-LF showed values of 28 and 36%, respectively. Using statistical analysis, the binding affinity for MC-LR was significantly different from the values for other derivatives ( p-value << 0.01 as determined by t-test).

Example  5: QD - Apta Sensor  And ELISA Assay  Intracellular MC- in Pure Cyanobacteria Cultures LR  analysis

Microcystis aeruginosa ( M. aeruginosa ) strain UTEX 2388, known as the producer of hepatotoxin, was obtained from the Culture Collection of Algae of the University of Texas, USA. Bacteria were inoculated into 250 mL Erlenmeyer flasks containing modified Bold 3N medium from which 100 mL of soil water was removed. While stirring the bacteria at 100 rpm (Orbital shaker, DAIHAN Scientific, Gangwon, Korea), under continuous illumination (20,000 lux, 30 W, SL230D, City ELG, Incheon, Korea), 265 hours at room temperature (about 11 days). Culture broth was periodically taken from the flask (0, 18.5, 42.5, 74.5, 96, 114, 139, 163, 186.5, 210.5, 235, and 265 hours) while incubating. Cell growth of M. aeruginosa was determined by analyzing the absorbance of the culture at 680 nm with a SpectraMax M2 spectrofluorometer. For intracellular MC-LR extraction, aliquots of M. aeruginosa culture (1.5 mL) were collected in 1.7 mL vials by centrifugation (Centrifuge 5418, Eppendorf, Hamburg, Germany) for 3 minutes at 14,000 rpm. The collected cells were suspended in 1 mL of distilled water. Place the vial on ice and cycle three times (1 cycle: 30 sec on and 30 sec cool down) to an XL-2000 ultrasonic crusher (ultrasonic dismembrator, Qsonica, Newtown, CT, USA) with a P-3 microprobe at 10 W Ultrasonicated. Lysed cells were removed by centrifugation in the same manner. The supernatant was filtered with a 25 mm nylon syringe filter (0.45 μm, CHMLAB, Barcelona, Spain). The obtained extract was used for further MC-LR analysis using QD-Apta sensor and ELISA. QD-Apta sensor was prepared according to the experimental procedure in the sensitivity check. M. aeruginosa culture extract was added to the plate and incubated for 6 hours at room temperature with stirring at 1,200 rpm in MixMate. After incubation, fluorescence of QD ₅₂₅ and FRET-based PoPo3 dyes was measured using SpectraMax M2 spectrofluorometer. Relative normalized fluorescence was determined according to equation (2) above. All experiments were performed three times. MC-LR concentrations were calculated from the calibration curves plotting the relative normalized fluorescence with MC-LR concentrations (FIG. 5 b ). For comparison, M. aeruginosa culture extracts were also analyzed with an ELISA assay kit (Microcystin-LR ELISA Kit, Abnova, Taipei City, Taiwan) according to the manufacturer's instructions. MC-LR concentrations in extracts were estimated from standard curves obtained from commercial ELISA kits (0.1-2.5 ng / mL, FIG. 7). All experiments were performed three times.

Pure cyanobacteria cultures and environmental water samples were used to confirm the applicability of the FRET-based QD-Apta sensor. First, the intracellular MC-LR concentration of M. aeruginosa culture medium was measured quantitatively using FRET-based QD-apta sensor according to cell growth, and also analyzed using a conventional ELISA assay for comparison.

As shown in FIG. 8A, M. aeruginosa cells were grown after 18.5 hours of lag period. Following exponential growth in the early stages, the stationary growth phase began in about 139 hours of incubation. Maximum specific growth rate (μ _max ) reached 0.0199 ± 0.0002 h ⁻¹ (FIG. 8B). Each MC-LR concentration was measured simultaneously via both FRET based QD-Apta sensor and ELISA assay (FIG. 8A). The ELISA assay was unable to quantitate MC-LR during 42.5 hours of induction and early-exponential growth, whereas FRET-based QD-apta sensors were detected with 0.02-0.15 ng / mL-cyanobacterial cultures. This may be due to the superiority (eg, lower LOD) of the FRET based QD-Apta sensor to MC-LR detection. As shown in FIG. 7 and FIG. 5B, the assay range of ELISA assay was 0.1-2.5 ng / mL for MC-LR, whereas the assay range of QD-Apta sensor was 10 ⁻⁴ to 10 ² ng / mL. Both methods showed lower intracellular MC-LR concentrations of about 3.5 ng / mL-cyanobacterial cultures up to 139 hours of exponential phase, then 12.7-15.8 ng / mL- in the initial stationary phase from 139 hours to 210.5 hours. There was a rapid increase in cyanobacteria cultures. However, intracellular MC-LR concentrations then decreased significantly from 1.9 to 4.2 ng / mL-cyanobacterial cultures. The decrease during the late exponential phase appears to be due to cell lysis, thus releasing MC-LR molecules of 10.8 to 11.6 ng / mL-cyanobacterial culture from inside the cell into the culture medium. As a result, the measured intracellular MC-LR concentration was much lower than the maximum concentration. This is consistent with previous studies. As more cells were lysed at later stages of cyanohydration, extracellular MC-LR became more abundant in water. Intracellular MC-LR concentration measured by FRET based QD-Apta sensor showed a significant positive correlation with the results by ELISA assay (FIG. 8C). This represents the Pearson's correlation coefficient ( p -value << 0.01) with r = 0.981 between the two variables.

Example  6: QD - The apta sensor  MC in the actual water sample through LR  analysis

Environmental water samples were collected from the Han River in August 2016. Six points (two negative control points of NC1 and NC2 and a study point of S1, S2, S3, and S4) were selected for water sampling. Site map and water quality information are shown in Figure 9 and Table 3, respectively. The algal densities of S1, S2, S3, and S4 were 8.6 × 10 ³ , 6.5 × 10 ³ , 2.7 × 10 ⁵ , and 6.6 × 10 ⁷ cells / mL, respectively. Two positive controls (PC1 and PC2) were also prepared by adding small amounts of M. aeruginosa culture to NC1 and NC2. Aliquots (1 mL) of water samples were transferred to 1.5 mL vials. The vial was placed on ice and sonicated three times using an XL-2000 ultrasonic crusher in the same manner as in the above example. After sonication, the water sample was filtered through a 25 mm nylon syringe filter (0.45 μm). Filtrates (filtrates, 20 μL) were used for further analysis of MC-LR using QD-Apta sensor and ELISA assay. As depicted in the MC-LR analysis in M. aeruginosa cultures, QD-AptaSensor and ELISA assays were performed. MC-LR concentrations in environmental samples of environmental eutrophic water were estimated using standard curves of QD-Apta sensors and ELISA assays (FIGS. 5B and 7). All experiments were performed three times.

sample location Latitude/
Hardness Water temperature
(℃) pH DO
(mg / L) BOD
(mg / L) COD
(mg / L) SS
(mg / L) TN
(mg / L) TP
(mg / L) NC1 Seoul Seocho 37.47 /
127.03 29.2 7.9 7.9 1.5 5.0 6.8 5.02 0.317 NC2 Gyeonggi Anyang 37.39 /
126.97 28.4 7.6 4.5 3.8 7.7 5.6 7.73 0.040 S1 Seoul Gangdong 37.54 /
127.11 28.2 7.9 8.3 2.0 4.7 5.5 2.37 0.036 S2 Seongdong, Seoul 37.53 /
127.03 29.1 8.1 6.7 0.8 3.6 3.4 2.44 0.031 S3 Yongsan, Seoul 37.52 /
126.96 29.6 7.4 4.2 1.5 4.3 6.8 3.45 0.084 S4 Seoul Yeongdeungpo 37.55 /
126.89 30 7.7 5.1 1.4 5.4 11.6 3.19 0.103

We also evaluated the performance of the FRET-based QD-Apta sensor using real environmental water samples. As shown in FIG. 10A, the target toxin, MC-LR, was only detected in samples of S3, S4, PC1, and PC2 at concentrations of 1.0, 7.2, 2.8, and 2.3 ng / mL-water, respectively. For comparison, these samples were analyzed by conventional ELISA assays, and the resulting MC-LRs were identified at concentrations of 0.2, 5.3, 3.7, and 2.3 ng / mL-water, respectively. Both of these assays measured the concentration of MC-LR without isolation and purification of cyanotoxins in eutrophic water samples. The MC-LR concentration measured via the QD-Apta sensor was similar to our previous study, which quantifies the microcystin synthetase D ( mcyD ) gene in the same water sample (Environ. Sci. Technol., 2018, 52: 1375-1385). S3, S4, PC1, and PC2 samples showed 3.5 × 10 ³ , 5.6 × 10 ⁶ , 1.2 × 10 ⁵ , and 2.0 × 10 ⁵ mcyD gene copy number / mL-water, respectively. MC-LR concentration analyzed by QD-Apta sensor for ELISA assay showed a strong positive linear correlation (FIG. 10B). This represented a Pearson correlation coefficient of r = 0.862 ( p -value << 0.01).

The FRET-based QD-Apta sensor developed in the present invention was suitable for detecting MC-LR in both laboratory and real water samples. This has a performance comparable to existing ELISA assays. More importantly, the MC-LR concentration in laboratory cultures exhibits a wide range of 0.02 to 15.8 ng / mL during cyanobacteria growth. As mentioned above, existing ELISA assays have a narrow assay range of 0.1 to 2.5 ng / mL, thus requiring preconcentration or dilution of the sample prior to analysis. On the other hand, FRET-based QD-Apta sensors can perform MC-LR analysis without additional preprocessing. This indicates that the FRET-based QD-Apta sensor developed in the present invention is a suitable means to enable highly sensitive and selective in-situ MC-LR detection.

Comparative example  1: using organic phosphor as receptor QD - Apta Sensor  Based detection method

In the detection method by FRET using a QD-apta sensor, in order to confirm the effect by the combination of a fluorescent donor and a receptor, a complex similar to the QD-apta sensor of the present invention is prepared, but the maximum emission at 565 nm as a quantum dot. An aptamer that specifically binds to DEHP ((di-2-ethylhexyl) phthalate) was bound thereto using QD ₅₆₅ , which represents an organic phosphor, carboxy-X-rhodamine (carboxy-X). -rhodamine (ROX) was covalently bound to the aptamer. The complex prepared as described above was detected while contacting with various concentrations of DEHP, and the results are shown in FIG. 11. As shown in Figure 11, when using a complex covalently bonded to the organic phosphor in the aptamer as a fluorescent receptor, the same pattern as the control (control, that is, only the solvent containing no DEHP) at the total concentration shows the same pattern It was. This indicates that the detection of the sample may not be possible even if the combination of the wavelength-matched fluorescent donor and the receptor to generate FRET.

<110> Ewha University-Industry Collaboration Foundation <120> Composition for detecting microcystin-LR comprising magnetic beads,          quantum dots and DNA intercalating dyes <130> KPA180400-KR <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> AN6 aptamer <400> 1 ggcgccaaac aggaccacca tgacaattac ccataccacc tcattatgcc ccatctccgc 60

Claims (14)

Magnetic beads;
Two or more quantum dots (QD ₅₂₅ ), having a maximum emission wavelength at 525 nm, coupled to the surface of the magnetic beads;
DNA aptamers that specifically bind to microcystin-LR, bound on quantum dots; And
PoPo3 dye (2-((E) -3- [1- (3-[[3- (dimethyl (3- [4-[(E) -3- (3-methyl)) having a maximum absorption wavelength at 533 nm. -1,3-benzoxazol-3-ium-2-yl) -2-propenylidene] -1 (4H) -pyridinyl] propyl) ammonio) propyl] (dimethyl) ammonio] propyl) -4 (1H) -pyridinylidene]- Microcystine-LR detection composition comprising 1-propenyl) -3-methyl-1,3-benzoxazol-3-ium tetraiodide).
The method of claim 1,
DNA aptamer specifically binding to the microcystin-LR (microcystin-LR) is a composition consisting of an aptamer consisting of the nucleotide sequence of SEQ ID NO: 1.
delete The method of claim 1,
Wherein said PoPo3 dye is intercalated in the DNA aptamer in the absence of microcystine-LR and detached upon contact with microcystine-LR.
The method of claim 1,
Wherein the composition exhibits an emission spectrum in the region consistent with the fluorescence spectrum of the PoPo3 dye when it is excited by irradiation with light of 500 to 550 nm wavelength in the absence of microcystine-LR.
The method of claim 1,
Wherein said composition exhibits an emission spectrum of quantum dot QD ₅₂₅ when excited by irradiation with light at a wavelength of 500-550 nm upon contact with microcystine-LR.
The method of claim 1,
The surface of the magnetic bead and one end of the aptamer is modified with an amine group, the surface of the quantum dot QD ₅₂₅ is modified with a carboxyl group, or the surface of the magnetic bead and one end of the aptamer is modified with a carboxyl group, the surface of the quantum dot QD ₅₂₅ is A composition modified with an amine group.
The method of claim 7, wherein
The magnetic bead surface, the quantum dot, and the quantum dot surface and the aptamer are N-hydroxysuccinimide (NHS) and ethylcarbodiimide hydrochloride (EDC) through a carboxyl group and an amine group included at each end. The composition is bound by reaction with.
Microcystine-LR detection kit comprising the composition of claim 1.
The method of claim 9,
The composition is N-hydroxysuccinimide (NHS) in the form of a precursor of a magnetic bead modified with an amine group, aptamer modified with an amine group at one end, and a QD ₅₂₅ surface modified with a carboxyl group. And ethylcarbodiimide hydrochloride (EDC).
Water samples suspected of being contaminated with microcystine-LR were magnetic beads; At least two quantum dots having a maximum emission wavelength at 525 nm, coupled to the surface of the magnetic beads; DNA aptamers that specifically bind to microcystine-LR bound on the quantum dots; And a first step of contacting with a probe comprising a PoPo3 dye inserted into the DNA aptamer; And
Irradiating light of 500 to 550 nm wavelength and measuring the emission spectrum of the PoPo3 dye; Microcystine-LR detection method in a water sample comprising a.
The method of claim 11,
Magnetic beads prior to the first step; At least two quantum dots having a maximum emission wavelength at 525 nm, coupled to the surface of the magnetic beads; DNA aptamers that specifically bind to microcystine-LR bound on the quantum dots; And irradiating light having a wavelength of 500 to 550 nm to a probe including the PoPo3 dye inserted into the DNA aptamer and measuring the emission spectrum of the PoPo3 dye. Way.
The method of claim 12,
Comparing the spectrum measured from the second stage with the spectrum measured from the first stage to determine that the water sample contains microcystine-LR when the emission signal of the spectrum measured from the second stage is lower. Microcystine-LR detection method further comprising the step.
The method of claim 13,
The method of detecting the microcystine-LR is to calculate the concentration of the microcystine-LR contained in the water sample from the reduced degree of the luminescence signal of the spectrum measured from the second step compared with the spectrum measured from the step 1-1. .

Images