KR20130104779A - Method for detecting contaminants from samples using quantum dot based competitive immunoassay and multiplexed flow cytometric readout - Google Patents

Abstract

PURPOSE: A method for detecting a contaminant from a sample by quantum dot-based competitive immunoassay and multiplex flow cytometry is provided to enhance sensitivity (reduction of LOD and increase of measurement range), to shorten analysis time, and to simplify operation, thereby quickly and easily diagnosing dangerous factors in the sample with high sensitivity. CONSTITUTION: A method for detecting a contaminant from a sample by quantum dot-based competitive immunoassay and multiplex flow cytometry comprises the steps of: preparing polystyrene bead-antigen competitive complexes in which each antigen is conjugated to polystyrene beads; preparing quantum dot probes in which each antibody is conjugated to quantum dots; applying the polystyrene bead-antigen competitive complexes to a sample solution to be analyzed; and adding the quantum dot probes; and analyzing the sample solution using a flow cytometry device. [Reference numerals] (1) Produce a sample solution; (2) Add polystyrene bead-antigen (PB/Ag) competitive complexes; (3) Add quantum dot detecting probe; (4) Competitive immune reaction; (5) Implement a flow cytometry; (AA) Pollutant (antigen) 1; (BB) Pollutant (antigen) 2; (CC) PB/Ag complex 1; (DD) PB/Ag complex 2; (EE) Quantum dot detecting probe 1; (FF) Quantum dot detecting probe 2; (GG) Quantum dot detecting probe combined PB/Ag complex; (HH) Laser; (II) Photoelectron multiplier (fluorescence channel 1); (JJ) Photoelectron multiplier (fluorescence channel 3); (KK) Collect data; (SS) Side Wall Scattering

Description

Method for detecting contaminants from samples using quantum dot based competitive immunoassay and multiplexed flow cytometric readout

The present invention relates to a method for multiple detection of contaminants from a sample using quantum dot based competitive immunoassays and multiple flow cytometry.

The detection of environmental pollutants is of great concern for drinking water-related sanitation, which is mainly associated with human exposure to microorganisms and chemical hazards. To ensure the safety of drinking water effectively, strict water quality standards and requirements must be met, which is a more powerful analysis for diagnosing environmental pollutants with high sensitivity, speed and ease of use while overcoming the disadvantages of conventional analytical techniques. A device is needed. Furthermore, real field analysis requires the ability to measure several specific target compounds in complex water samples simultaneously.

Currently, the two main methods for multiplex analysis are planar and bead-based alignment (Braeckmans, K .; De Smedt, SC; Leblans, M .; Pauwels, depending on the reaction site and method of identification of the analyte). R .; Demeester, J., Encoding microcarriers :. present and future technologies Nat Rev Drug Discov 2002, 1 , (6), 447-456). First, in planar alignment, target molecules react and code on a plane and are detected according to the exact position (x, y coordinate), such as a microchip. Next, in multiple assays, the reaction occurs on microspheres and the reaction is tracked according to the coded bead properties. Assays using beads have significant advantages over planar microarrays because of their fast molecular response and fast read performance (Deng, W .; Drozdowicz-Tomsia, K .; Jin, D .; Goldys, EM, Enhanced Flow). Cytometry-Based Bead Immunoassays Using Metal Nanostructures . Analytical Chemistry 2009, 81, (17), 7248-7255). This assay can simultaneously detect multiple target components in complex mixtures (multiplexing detection), which is used to label optically coded microbeads with fluorescently coated biomolecules to detect fluorescence (eg flow cytometers). It is possible to detect by.

Flow cytometry is a powerful technique for rapidly analyzing multiple properties of large numbers of cells and other microparticles in a single fluid stream (Soman, CP; Giorgio, TD, Quantum Dot Self-Assembly for Protein Detection with Sub-Picomolar Sensitivity. Langmuir 2008, 24 , (8), 4399-4404), a common technique in the field of biomedical research (ie oncology, immunology and toxicology), but (Hahn, MA; Keng, PC; Krauss, TD, Flow Cytometric Analysis To Detect Pathogens in Bacterial Cell Mixtures Using Semiconductor Quantum Dots. Analytical Chemistry 2008, 80 , (3), 864-872, relatively little research in other fields such as environment and microbiology (Anderson, GP; Lamar, JD; Charles, PT, Development of a Luminex Based Competitive Immunoassay for 2,4) , 6-Trinitrotoluene (TNT). Environmental Science & Technology 2007, 41 , (8), 2888-2893, etc.). Unlike other fluorescence analyzers, which measure the average spectral characteristics of an entire sample, flow cytometry can examine almost every individual particle in a sample, and allows for multiple properties such as light scattering (size and granularity) and fluorescence intensity at various wavelengths. Measure at the same time. Because of these advantages, flow cytometry can clearly distinguish specific individuals from multiparametric analyzes across the population.

Essentially in flow cytometry, fluorescent dyes (fluorescent markers or probes) conjugated with biomolecules specific for the target compound are used, which is necessary to recognize and quantify a particular cell population. Organic dyes are commonly used for this purpose, but there are some serious drawbacks and limitations when using multiple organic dyes simultaneously to analyze multiple targets; (1) Analyzing the narrow absorption spectrum of organic dyes requires auxiliary excitation sources such as multi-wavelength lasers or Fluorescence Resonance Energy Transfer (FRET) systems, which complicates the experimental setup and increases costs. (2) wide emission characteristics of organic dyes cause spectral overlap between fluorescent dyes, requiring additional color compensation and correction processes, and (3) low quantum efficiency (Chattopadhyay, PK; Price, DA; Harper, TF; Betts, MR; Yu, J .; Gostick, E .; Perfetto, SP; Goepfert, P .; Koup, RA; De Rosa, SC; Bruchez, MP; Roederer, M., Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nature Medicine 2006, 12 , (8), 972-977, etc.).

Therefore, research has been reported to use quantum dots (QDs), which are semiconductor nanoparticles instead of such fluorescent dyes, to improve the aforementioned problems (Chattopadhyay, PK; Perfetto, SP; Yu, J .; Roederer, M., The use of quantum dot nanocrystals in multicolor flow cytometry. Wiley Interdisciplinary Reviews : Nanomedicine and Nanobiotechnology 2010, 2 , (4), 334-348, etc.). Unlike organic dyes, QDs have a broad absorption spectrum, which eliminates the need for FRET inducers and can be used to construct experimental devices with just one excitation source. In addition, the emission spectrum (half width ~ 24-40 nm) of the QD is narrow and symmetrical, so there is almost no spectral overlap and no color correction is necessary. Furthermore, since QD has a high quantum yield and a high molar extinction coefficient (˜10-100 times that of organic dyes), the ability to distinguish a background signal from a dyed population, i.e., sensitivity, is improved.

Accordingly, the present invention provides a quantum dot-based competitive immunoassay combined with flow cytometry to detect several analytes simultaneously, thereby providing an apparatus for easily detecting environmental contaminants with high sensitivity and high speed.

In order to solve the above problems,

Preparing respective polystyrene bead-antigen competition complexes to which each antigen to be detected is bound to the polystyrene beads;

Preparing respective quantum dot detection probes in which quantum dots are bound to each of the antigens to be detected and each antibody capable of performing an antigen-antibody immune response;

Adding the polystyrene bead-antigen competition complexes to the sample solution to be analyzed;

Applying the quantum dot detection probes to the sample liquid to be analyzed; And

It provides a method for detecting contaminants in a sample using a quantum dot-based competitive immunoassay and multiple flow cytometry comprising the step of analyzing the sample solution in the flow cytometry.

According to one embodiment of the invention, the polystyrene bead-antigen competition complex may be formed by conjugation reaction of streptavidin coated polystyrene beads with biotinylated detection antigen.

According to another embodiment of the present invention, the quantum dot detection probes may be prepared by coating a monoclonal antibody against the antigen to be detected on the surface of the quantum dot.

According to another embodiment of the present invention, after applying the quantum dot detection probes to the sample liquid, the competition between the antigen to be detected in the sample liquid and the antibody bound to the quantum dot detection probe The method may further include allowing an antigen-antibody immune response and an antigen-antibody immune response between the antigen bound to the polystyrene bead-antigen competition complex and the antibody bound to the quantum dot detection probe to reach an equilibrium state.

According to another embodiment of the present invention, the flow cytometer may provide information about the size parameter of the electrical volume, the granularity of the side scattering parameter and the fluorescence parameter.

According to another embodiment of the present invention, each antigen to be detected may be microcystine-LR and benzo [a] pyrene.

According to another embodiment of the present invention, the quantum dots may be quantum dots emitting fluorescence at 525nm and quantum dots emitting fluorescence at 655nm.

According to another embodiment of the present invention, the quantum dot detection probe is a quantum dot detection probe attached to the microcystine-LR antibody to the quantum dot emitting fluorescence at 525nm and the benzo [ a] may be a quantum dot detection probe to which a pyrene antibody is attached.

According to the present invention, it is possible to improve sensitivity (reduce detection limit and extend measurement range), shorten analysis time and simplify operation, based on flow cytometry reading method having excellent throughput and throughput, Therefore, it is possible to easily detect multiple risk factors in a sample at the same time with high sensitivity and high speed.

1 is a schematic process diagram of a method for multiple detection of contaminants in accordance with the present invention.
FIG. 2 shows gated regions for polystyrene bead-antigen competition complexes and quantum dot detection probes, FIG. 2A shows histograms for electrical volume, FIG. 2B shows electrical volume in gated region 1 (inset), Density plots for side scattering and fluorescence histograms.
3 is a graph for the incubation time optimization process in a competitive immunoassay for the detection of microcystine-LR and benzo [a] pyrene.
4A (standard microcystine-LR) and 4b (standard benzo [a] pyrene) are graphs showing a calibration curve showing the relationship between QD fluorescence intensity and log of standard analyte concentration, and insets show antigen concentration and QD fluorescence intensity. It is a measurement range showing the linear relationship between them.
FIG. 5 is a confocal laser scanning microscopy image for visualization of a QD detection probe bound to a polystyrene bead competitor, in FIG. 5, (a) and (c) are pure polystyrene bead-antibody competition complexes and QD detection probes bound thereto A differential interference contrast mode image for the child, (b) and (d) are fluorescence mode images of the pure polystyrene bead-antibody competition complex and the QD detection probe bound thereto, (a + b) And (c + d) are the sum of a and b and the sum of c and d, respectively.
FIG. 6 is a graph showing a calibration curve showing the relationship between QD fluorescence intensity and log of standard analyte concentration in a sample in which microcystine-LR and benzo [a] pyrene are present simultaneously. FIG. Is a graph for microcystine-LR detection under FIG. 6B is a graph for benzo [a] pyrene detection in the presence of microcystine-LR.
FIG. 7 is _a graph showing the emission spectra of two QD525 / Ab _{microcystine- LR} and QD655 / Ab _{benzo [a] pyrene} detection probes, calculated as% fluorescence values detected and spilled over in the FL1 and FL3 channels, respectively.

In the present invention, specific immune responses are simultaneously performed using two sensing elements (antibody-coated QD detection probes and antigen-fixed polystyrene beads) with different colors for quantitative analysis based on flow cytometry. The two sensing elements respond in a competitive immunity to each target compound. Depending on the target material, microbeads specifically stained with different colors of QD are simultaneously characterized and interpreted using flow cytometry, where flow cytometry selectively discriminates specifically targeted microbeads and displays fluorescence. It can be measured. The fluorescence intensity of each QD recorded on a particular detector directly provides qualitative and quantitative information about each target molecule.

The method for detecting contaminants in a sample using quantum dot-based competitive immunoassay and multiple flow cytometry according to the present invention comprises the steps of preparing respective polystyrene bead-antigen competition complexes in which each antigen is bound to polystyrene beads. ; Preparing respective quantum dot detection probes in which quantum dots are bound to each of the antigens to be detected and each antibody capable of performing an antigen-antibody immune response; Adding the polystyrene bead-antigen competition complexes to the sample solution to be analyzed; Applying the quantum dot detection probes to the sample liquid to be analyzed; And analyzing the sample liquid to be analyzed in a flow cytometry apparatus.

1 shows a schematic process diagram of a method for detecting contaminants according to the present invention. FIG. 1 shows a method for an analyte sample (FIG. 1, step 1) comprising two kinds of antigens (indicated by yellow spheres and purple spheres in the drawing). Referring to FIG. A polystyrene bead-antigen competition complex will be prepared that will undergo a competitive immune response with the substance (antigen). In addition, a quantum dot detection probe is prepared in which a quantum dot is combined with an antibody capable of performing an antigen-antibody immune reaction with an antigen in a contaminant (antigen) or a polystyrene bead-antigen competition complex in the sample to be analyzed. The polystyrene bead-antigen competition complex thus prepared is added to the sample to be analyzed (FIG. 1, step 2). Next, the prepared quantum dot detection probe is added to the sample to be analyzed (FIG. 1, step 3). Thus, when the polystyrene bead-antigen competition complex and the quantum dot detection probe are added to the sample to be analyzed, the antigen-antibody reaction between the contaminants (antigens) and the quantum dot detection probe in the sample to be analyzed and the polystyrene bead-antigen competition Antigen-antibody reactions between the antigens of the complex and the quantum dot detection probes are made competitive (FIG. 1, step 4). Thereafter, the sample to be analyzed is finally analyzed in a flow cytometry device, so that contaminants (antigens) in the sample to be analyzed can be qualitatively and quantitatively analyzed.

The polystyrene bead-antigen competition complex may be prepared by various methods of attaching an antigen to be detected on polystyrene beads, but is not limited thereto, for example, biotinylated with streptavidin coated polystyrene beads. It can manufacture by the conjugation reaction of a detection antigen.

In addition, the quantum dot detection probes can be prepared by coating a monoclonal antibody against the antigen to be detected on the surface of the quantum dot.

On the other hand, after the polystyrene bead-antigen competition complex and the quantum dot detection probes are added to the sample solution to be analyzed, the antibody bound to the quantum dot detection probe is the antigen-competitive with the detection antigen and the polystyrene bead-antigen competition complex- The antibody immune response may further include the step of allowing the antigen-antibody immune response to reach an equilibrium for a predetermined time (left after FIG. 1 and step 4).

In the present invention, a variety of information on the contaminants in the sample to be analyzed is provided for the sample to be transferred to the flow cytometer. Non-limiting examples of such information include information about the size parameter of the electrical volume, the granularity discrimination parameter of lateral scattering, and the fluorescence parameter.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are merely to aid the understanding of the present invention, and the scope of the present invention is not limited to the following Examples.

Example

Hereinafter, in the following examples, two examples of environmental risk factors in drinking water are substances derived from microorganisms (microcystin-LR, which is a cyanobacteria toxin), and substances derived from compounds (benzo [a] pyrene, which is a polycyclic aromatic hydrocarbon (PAH)). The method according to the invention was carried out using.

Qdot  ( QD ) / Antibody ( Ab ) detection Probe  Produce

Monoclonals for microcystine-LR (microcystine-LR, MC10E7, Enzo Life Sciences) and benzo [a] pyrene (22F12, Munich, Germany) to make color markers (green or red) for immunological recognition Antibodies (mAbs) were coated on the surface of QD (Qdot525 and QD655 antibody conjugation kit, Invitrogen), respectively, according to manufacturer's instructions. The binding capacity of the synthesized QD525 / Ab _{microcystine- LR} detection probe was already estimated to be 10 microcystine-LR molecules per probe (Yu, HW; Lee, J .; Kim, S .; Kim, SJ; Kim, IS). , Photoelectronic characterization of IgG antibody molecule-quantum dot hybrid as biosensing probe.Nanotechnology 2010, 21 , (42), 425501-425511), it is estimated that eight antigens can be conjugated with the QD655 / Ab _{benzo [a] pyrene} detection probe (Invitrogen).

polystyrene Bead  ( PB ) / Antigen ( Ag Competitor Synthesis

Each antigen (microcystine-LR or benzo [a] pyrene derivatives) is added to polystyrene beads through a contiguous two-step reaction, i.e., biotinylation of each antigen and conjugation of biotinylated molecules to streptavidin-coated PB surfaces. PB / Ag competitors were made by binding. Next, 0.30 μmol of the microcystine-LR (Alexis) and benzo [a] pyrene derivatives (c- (10-benzo [a] pyrenyl) -butyric acid, B [a] P-10-C4, Professor Xideel A) 0.45 μmol N-hydroxysuccinimide (NHS) and 0.45 μmol dissolved in anhydrous dimethylsulfoxide (DMSO) / acetonitrile (25: 75% v / v). Mix with dicyclohexylcarbodiimide (DCC) in a desiccator for 24 hours. Thereafter, the activated NHS ester of the carboxylic acid was reacted with a primary amine group of 0.60 μmol EZ-linked amine-PEG3-biotin (thermo Scientific) for 21 hours, and then incubated with triethylamine (0.45 μmol) for 3 hours to remain. The reactivity of the unreacted amine-reactor was removed. To minimize protein polymerization the ratio of the reagents was chosen using an excess molar amount of amine-PEG3-biotin and a limited amount of DCC. In addition, a biotin-modified molecular suspension (functionalized) of 1 mL of streptavidin-coated polystyrene beads (Sphero streptavidin polystyrene particles (5.0-5.9 μm), spherotech) corresponding to a biotin binding capacity of 0.74 nmol was prepared. Cultured for 24 hours with constant mixing with 400 times the molar amount compared to the biotin binding capacity of the microbeads. Unbound material was then removed from the synthesized PB competitor by centrifuging several times with PBS buffer using 0.45 μm filter tubes (Watman). The final complex was then resuspended and stored in 0.02% sodium azide / PBS as a preservative buffer at 4 ° C. before use.

Optimization of Competitive Immune Response

Factors affecting the competitive immune response were optimized to improve the sensitivity and analysis time. Reagent concentrations of QD detection probes (2.5 × 10 ⁻⁸ to 2.0 × 10 ⁻⁷ M for QD525 / Ab _{microcystine- LR} , to identify optimal experimental conditions for competitive immunological recognition and further generation of QD fluorescence signals) 5.0 × 10 ^-8 to 4.0 × 10 ^-7 M for QD655 / Ab _{benzo [a] pyrene} , reagent concentrations of PB competitor (2.4 × 10 ^-14 to 1.9 × 10 ^-13 M) and incubation time (10-120 Minutes).

Immunological Perception: Competitive Immunoassay

It was prepared from 50 μg / L range streak dilution method using a 100% methanol standard Microcystin -LR and benzo [a] pyrene 10 ^-3, respectively in order to analyze the environmental ^pollutant-2 10 μg / L and ^10-4 . Each analyte was detected for 30 minutes at room temperature for microcystine-LR (9.5 × 10 ⁻¹⁴ M PB / microcystine-LR and 1.0 × 10 ⁻⁷ M QD525 / Ab _{microcystine- LR} ) or benzo [a] pyrene and cultured using a detection (2.4 × 10 ^-14 M PB / benzo [a] pyrene and 2.0 × 10 ^-7 M QD655 / _{Ab-benzo [a] P} of the _alkylene) sensing elements set of 20 μl. After the completion of the competitive immune response, as a final analysis step, fluorescence measurements of each QD without undergoing a washing step were performed using a flow cytometer.

Signal conversion: Flow cell  Analysis method

Each analyte sample alone or a mixture thereof was analyzed with a Cell Lab Quanta SC flow cytometer (Beckman Coulter) equipped with a 22 mW 488 nm excitation laser. A single microbead carrier fluid: 10 ⁵ using (sheath fluid iso-diluent, NPE, Inc.) by suspending the particles in a density of 10 ⁷ / ml, the narrow hole and the tubes of the flow cytometer was not blocked. Prior to each experiment, flow-check fluorospheres (Beckman Coulter) were used for quality control (QC) for 488 nm laser applications, allowing the device to operate accurately and accurately. 100,000 particles exceeding 4 μm in diameter (EV channel = 100) set by the lower level discriminator (LLD) of the Electronic Volume (EV) were counted and analyzed. By setting the channel for the EV parameters, individuals below the setpoint were excluded and the signals caused by unreacted QD / Ab detection probes, antigens, their conjugates and impurities were removed. Flow cytometer EV channel axis (μm ³ ) was converted to particle size (μm) with 5 μm calibration beads (Coulter CC size standard L5, Beckman Coulter). The flow rate of the sample was controlled to keep the count rate constant (600-700) during the sample experiment. It is important to set the gain, PMT voltage and discrimination values appropriately to distinguish the EUT from background noise. The gain values of EV and side scatter (SS) were 5.23 and 3.40, respectively. Photomultiplier tube (PMT) voltages for two-color fluorescence measurements of green (fluorescent channels 1, F1) and red (fluorescent channels 3, F3) were also set to 6.04 and 5.98, respectively. For quantitative analysis of each antigen (microcystine-LR and benzo [a] pyrene), flow cytometry data of four parameters (EV, SS, FL1 and FL3) were collected.

Results and review

Flow cell  In the method Gating  ( gating Method and data interpretation

Microcystine-LR and benzo [a] pyrene are relatively small molecules having a molecular weight of only 995.17 g / mol (C ₄₉ H ₇₄ N ₁₀ O ₁₂ ) and 339.41 g / mol (C ₂₄ O ₂ H ₁₉ ), respectively. Sandwich immunoassays using heavy antibodies are difficult to apply due to steric hindrance of binding sites. Thus, in the present invention, a competitive immunoassay was constructed to recognize small molecules using a single antibody. In the present invention two main sensing elements (Ab-coated QD detection probes and antigen-fixed PB competitors) were introduced to compete with the target contaminants. The purpose of an antibody coated QD detection probe is to provide (1) immunological binding to specifically recognize the antigen of interest and (2) optical signal generation. The antigen-immobilized PB competitor also has two functions: (1) to compete with the analyte of interest in binding to a defined binding site of the solid carrier and the QD detection probe, and (2) for subsequent flow cytometry. There is a size determination function.

First, in the present invention, specific immunological responses are simultaneously performed using two sensing elements (QD / Ab detection probes and PB / Ag competitors) having different colors for quantitative analysis based on flow cytometry. The two sensing elements respond in a competitive immunity to each target compound. Depending on the target material, microbeads specifically stained with different colors of QD are simultaneously characterized and interpreted using flow cytometry, where flow cytometry selectively discriminates specifically targeted microbeads and displays fluorescence. It can be measured. The fluorescence intensity of each QD recorded on a particular detector directly provides qualitative and quantitative information about each target molecule.

After hydrodynamic focusing of the injected sample to be analyzed by the flow cytometer, coded microbeads are passed through the beam one by one after each immune response. Light scattering or fluorescence emission can be measured by three factors such as sizing parameter of electronic volume (EV), granularity differentiating parameter of side scatter (SS) and fluorescence parameter (FL). It is measured with two parameters, which thus provide information about the unique properties of the particles themselves. Analysis of the data obtained from the flow cytometer is performed by distinguishing the microbeads from non-bead-like individuals (unreacted QD / Ab detection probes, antigens, conjugates and impurities thereof), which is a microbead diameter of 5.04 μm. It is mainly through the EV having a value equal to or greater than the EV channel 100 corresponding to (Fig. 2A). As a more selective method of defining a population of interest (POI), the combination of EV and SS parameters is a simple, fast and effective method for characterizing QD-labeled PB competitors in heterogeneous samples. Can be. As shown in FIG. 2B, the conjugate with the PB competitor and its QD detection probe (high EV and high SS) using the EV / SS characteristics, reactants that are not bound to the surface of the PB competitor (low EV and low SS). Is clearly distinguished from. The desired POI represents the microbead assembly in the density graph of EV / SS, which is gated as region 1 (R1) for the positive data set, but the negative population (region 2, R2) was removed by the electron gate function. This electronic discrimination process is essential for conventional immunoassays but can replace time consuming washing steps. Once R1 selectively gates the microbeads of interest among the various sets of EV / SS plots, at the same time the dichroic fluorescence intensity is measured with a single excitation energy source. And it is recorded separately in different FL channels according to the colors of the fluorescence detection probe for quantification of each analyte. Fluorescence histogram (inset graph in FIG. 2B) shows the number of detections for relative fluorescence intensity, which is negative population (PB / Ag competitor alone as dye negative control), positive population (QD / Ab detection probe conjugated with PB / Ag competitor (I) The fluorescence intensity of For quantitative analysis, each fluorescence intensity of the green PB competitor for microcystine-LR and the red PB competitor for benzo [a] pyrene is collected and expressed as mean fluorescence intensity (MFI). As a result, it was found that the optical signal generated from the QD, that is, the ratio of competition to the PB competitor, was inversely proportional to the concentration of the microcystine-LR or benzo [a] pyrene analyte in the sample.

Optimization of competitive immunoassays: concentration of detection elements and response time

Once the assay configuration and signal transduction methods are established, the goal of optimizing immunoassays is to improve sensitivity and reduce assay time. ) And analytical conditions (concentration of the sensing element and incubation time). Sensitivity index (B / B _o ) described as the ratio between the optical signal (fluorescence) (B) obtained in the presence of the analyte and the signal (B _o ) in the absence of the analyte to determine the optimum experimental conditions. ) Was introduced. For further analysis, the optimal factor was selected that gives the lowest sensitivity index with the largest slope.

To optimize the best PB competitor and QD detection probe concentrations for each target analyte to improve assay sensitivity, as shown in Tables 1 and 2, tiled through serial 2-fold dilutions for various combinations. A titration experiment was conducted. On the basis of the sensitivity index evaluated as suitable for the desired measurement range in practical use, the most suitable QD detection probe and PB competitor concentrations were selected as follows: pair of sensing elements for microcystine-LR (QD525 / Ab _{microcystine-). LR} detection probe = 100 nM and PB / microcystine-LR competitor = 95 fM) and pairs for benzo [a] pyrene (QD655 / Ab _{benzo [a] pyrene} detection probe = 200 nM and PB / benzo [a] Pyrene competitor = 24 fM).

PB / Microcystin-LR Competitor (fM) QD525 / Ab _{Microcystine- LR} Detection Probe (nM ) 200 100 50 25 190 1.09 (4.45) ^b 0.98 (5.35) 0.92 (5.41) 0.89 (5.68) 95 0.99 (1.56) 0.82 (0.89) 0.83 (2.55) 0.88 (6.06) 48 0.94 (3.08) 0.92 (1.09) 0.85 (4.13) 0.89 (5.54) 24 0.96 (2.50) 0.84 (0.69) 0.87 (4.47) 0.82 (6.31)

PB / benzo [a] pyrene competitor (fM) QD655 / Ab _{Benzo [a] pyrene} Detection Probe (nM) 400 200 100 50 190 0.97 (8.23) 1.01 (7.32) 1.02 (6.86) 0.98 (3.76) 95 0.95 (4.21) 0.94 (8.34) 0.95 (6.32) 0.85 (8.31) 48 0.92 (2.17) 0.89 (4.51) 0.88 (6.82) 0.85 (2.35) 24 0.87 (7.43) 0.82 (4.87) 0.86 (9.30) 0.84 (7.44)

The injection concentration chosen is considered suitable to achieve optimal sensitivity because this set maintains an equilibrium between the antigen-antibody response equilibrium and the QD fluorescence generation and thus was used for further measurement of each analyte. . Since the total assay time is determined by the incubation time, the effect of time on competitive binding was investigated over a range of 10-120 minutes with the reagent concentration fixed at the above mentioned values. As shown in FIG. 3, a 30 minute incubation time corresponding to the minimum sensitivity index was selected as the most effective for competitive immunoassay based on MFI. The incubation time appeared to be insufficient for the QD detection probe to compete competitively with the analyte and the PB competitor until 30 minutes later. There is a tendency to move upward. Unlike planar assays, this fast binding rate allows antigen-antibody reactions to reach a faster equilibrium based on suspended bead assays, which results in greater surface area and increased diffusion distance to react with active biomolecules per volume of QD and PB particles. Because it becomes short.

Of a single analyte Flow cell  analysis

Once the general assay parameters are optimized, 10 ^-3 to 10 ² μg / L microcystine-LR and 10 ^-4 to 50 μg / to perform measurements of a single analyte using a competitive fluorescence immunoassay combined with flow cytometry. Each analyte standard solution in the L benzo [a] pyrene range was measured. Competitive immunological staining of the analytes using the QD525 / Ab _{microcystine- LR} and QD655 / Ab _{benzo [a] pyrene} detection probes to provide a green PB competitor for microcystine-LR and red for benzo [a] pyrene Fluorescence intensity (MFI) for the PB competitor at was measured at gated R1. 4 shows a calibration curve showing the relationship between the QD fluorescence intensity and the logarithm of the standard analyte concentration. The intensity of green (EL1) and red (FL3) fluorescence emitted from QD bound to microbeads was inversely proportional to the concentrations of microcystine-LR and benzo [a] pyrene in a single analyte sample, respectively. In order to confirm the sensitivity and reliability of the present invention, detection limits (LOD), quantitative limits (LOQ) and linear limits (LOL) were identified on standard curves. For single analyte measurements with microcystine-LR (WHO-presented drinking water rule <1 μg / L), the measurement range from LOQ (S / N = 10) to LOL is in the range of 0.36-30 μg / L (expressed as a percentage) One relative standard deviation (% RSD) was obtained from 1.1-8.5%); LOD (S / N = 3) was calculated to be 0.08 μg / L. For single analyte measurements of benzo [a] pyrene (the drinking water regulation presented by the European Council <10 ng / L), the optical signal is linear in the range 0.097-10 μg / L (% RSD is 1.3-12.7%), LOD was 0.044 μg / L. In contrast to microbead based fluorescence measurements using a spectrophotometer in our previous study (Yu, H.-W .; Jang, A .; Kim, LH; Kim, S.-J .; Kim, IS, Bead- Based Competitive Fluorescence Immunoassay for Sensitive and Rapid Diagnosis of Cyanotoxin Risk in Drinking Water. Environmental Science & Technology 2011, 45 , (18), 7804-7811), the assays of the present invention exhibit significantly higher sensitivity (wideer measuring ranges and slopes) because of the higher resolution and sensitivity associated with the measurement of individual particles in flow cytometry. Ability to distinguish light scattering and fluorescence).

Simultaneous for two analytes Flow cell  analysis

QD525 / Ab or QD655 / Ab detection probes were selectively bound to each target antigen-coated PB competitor via a competitive immune response in the presence of microcystine-LR and benzo [a] pyrene analytes in the same vessel. In order to simultaneously detect several fluorescent signals in a mixed sample, it is necessary to accurately read the optical signals in consideration of the possibility of spectral overlap between the fluorescent dyes (Chattopadhyay, PK; Price, DA; Harper, TF; Betts, MR; Yu, J .; Gostick, E .; Perfetto, SP; Goepfert, P .; Koup, RA; De Rosa, SC; Bruchez, MP;. Roederer, M., Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry Nature Medicine 2006, 12 , (8), 972-977). This fluorescence superposition is corrected through a process of subtracting the so-called compensation by estimating the magnitude of the spillover fluorescence as a fraction of the fluorescence measured at the main detector. The process is repeated for all channels until the signal in each channel represents the actual signal of the fluorescent dye to be detected. FIG. 7 shows the emission spectra of QD525 / Ab _{microcystine- LR} and QD655 / Ab _{benzo [a] pyrene} detection probes for analyzing spillover measured in FL1 and FL3 channels. Because their emission spectra are narrow and symmetrical, they show that QDs can be detected multiple times with minimal compensation, regardless of whether they are modified by biomolecules. Unlike organic fluorescent dyes, the spectral overlap between QDs is negligible, so that accurate positive signals can be measured as well as improved sensitivity. As shown in FIG. 5, QD-based competitive fluorescence immunoassay for simultaneous detection of two analytes could be achieved using different sets of sensing elements, PB / Ag competitor and QD / Ab detection probe. Each microbead encoded with a specific QD / Ab detection probe is formed by selective competition in the presence of two analytes. Quantitative information is provided through clear determination and fluorescence signal measurement by flow cytometry thereafter (see FIG. 6). Standard calibration curves for microcystine-LR or benzo [a] pyrene analytes are not significantly different from single analyte assays. Fluorescence signals generated from microbeads specific to the analyte decreased with increasing concentration of each antigen in the sample, regardless of the presence of other target analytes. However, the LOD and sensitivity of these assays may be slightly altered by fluorescence signal interference generated during the mixed immunoassay process. Nevertheless, it can be seen that QD-based flow cytometry in combination with a competitive immunoassay according to the present invention can simultaneously detect several different analytes.

Analytical Validation of Matrix Interference Effects

In order to confirm the performance of the present invention, the recovery of several water samples with potential interferences should be considered, which is caused by different types of water component variability. Therefore, the evaluation of this matrix effect is an important verification step to provide reliable data on environmental risk factors dissolved in water. In this example, the recovery rates of five water samples in the surrounding environment were evaluated by spiking standard microcystine-LR and benzo [a] pyrene as low, medium and high concentrations of internal analytes. Concentration ranges are those selected at regular concentration intervals within the measurement range of the method according to the invention (see Tables 3 and 4). Since the recovery is in the range of 90-110%, it can be seen that the present invention is not significantly affected by the nonspecific binding of the components suspended in water and thus can be applied to the direct measurement of environmental pollutants regardless of the type of water quality. .

Concentration of microcystine-LR (μg / L) % Recovery ^a Bottled water tap water River ^b Lake water ^c Sea water ^d Negative control 0 92.6 (13.7) 100.6 (10.9) 101.9 (12.4) 96.9 (14.7) 99.7 (15.4) that One 96.4 (11.3) 94.3 (12.0) 94.9 (13.4) 99.7 (18.7) 104.7 (9.3) medium 15 106.5 (9.3) 104.8 (10.2) 104.4 (10.3) 103.3 (7.6) 102.8 (10.6) The 30 92.6 (12.2) 97.5 (9.6) 97.0 (12.1) 96.3 (13.0) 94.1 (10.4)

Concentration of benzo [a] pyrene (ng / L) % Recovery ^a Bottled water tap water River ^b Lake water ^c Sea water ^d Negative control 0 104.0 (9.4) 91.5 (15.2) 100.6 (10.3) 96.4 (18.9) 90.5 (12.0) that 0.5 94.4 (12.9) 104.8 (8.5) 91.3 (8.1) 109.9 (5.8) 93.8 (11.0) medium 5 103.6 (17.4) 103.3 (8.8) 104.9 (17.8) 102.8 (16.5) 102.2 (8.4) The 10 95.4 (14.4) 107.2 (8.9) 109.8 (5.3) 104.9 (18.0) 110.3 (4.7)

a. % Recovery = (Measurement / Expected) × 100, all water samples were collected in winter and pretreated using a 0.2 μm syringe filter to rule out interference effects caused by particles or microbial cells. Figures in parentheses represent% CV based on three replicates.

b. The river was collected from Gwangjucheon (35 ° 09'N and 126 ° 50'E), Korea.

c. The lake water was collected from lakes (35 ° 13'N and 126 ° 50'E) located in Gwangju, South Korea.

d. Seawater was collected at points on the East Sea (35 ° 13'N and 129 ° 14'E).

As can be seen from the above examples, the assays established for the simultaneous detection of several environmental contaminants in the present invention have been successfully implemented by effectively incorporating QD-based competitive fluorescence immunoassays with flow cytometry readings. Compared to the conventional analytical methods, the combined technique of the present invention can achieve sensitivity enhancement (LOD reduction and measurement range extension), analysis time reduction and operation simplification. This high performance of the present invention is derived from three major improvements: suspension bead based competitive immune response, high throughput and rapid flow cytometry multiplex reading, and QD based optical signal amplification using a single excitation source. These distinctive features make the present invention very effective as a technique for diagnosing other environmental risk factors in drinking water with high sensitivity, quickly and easily. Furthermore, when combined with the small microfluidic flow cytometry of the present invention, it can be applied as a system for rapid and automatic screening of various toxic compounds simultaneously for the actual field evaluation of environmental samples.

Claims (8)

Preparing respective polystyrene bead-antigen competition complexes to which each antigen to be detected is bound to the polystyrene beads;
Preparing respective quantum dot detection probes in which quantum dots are bound to each of the antigens to be detected and each antibody capable of performing an antigen-antibody immune response;
Adding the polystyrene bead-antigen competition complexes to the sample solution to be analyzed;
Applying the quantum dot detection probes to the sample liquid to be analyzed; And
Method for detecting contaminants in a sample using a quantum dot-based competitive immunoassay and multiple flow cytometry comprising the step of analyzing the sample solution in the flow cytometry. The method of claim 1, wherein the polystyrene bead-antigen competition complex is formed by a conjugation reaction of a streptavidin-coated polystyrene bead and a biotinylated detection antigen, using a quantum dot-based competitive immunoassay and multiple flow cytometry. Method for detecting contaminants in a sample. The method according to claim 1, wherein the quantum dot detection probes are prepared by coating a monoclonal antibody against the antigen to be detected on the surface of the quantum dot, using a quantum dot-based competitive immunoassay and multiple flow cytometry. Method of detecting contaminants. The method of claim 1, wherein after applying the quantum dot detection probes to the sample solution, competitive antigen-antibody immunity between the antigen to be detected in the sample solution and the antibody bound to the quantum dot detection probe And allowing the antigen-antibody immune response of the antibody bound to the polystyrene bead-antigen competition complex to the antibody bound to the quantum dot detection probe to reach an equilibrium state. Method for detecting contaminants in a sample using competitive immunoassay and multiple flow cytometry. The sample using the quantum dot-based competitive immunoassay and multiple flow cytometry according to claim 1, wherein the flow cytometer provides information on an electrical volume size parameter, a side scattering granularity discrimination parameter, and a fluorescence parameter. Method of detecting heavy pollutants. The method of claim 1, wherein each antigen to be detected is microcystine-LR and benzo [a] pyrene. 4. The method of detecting contaminants in a sample using quantum dot-based competitive immunoassay and multiple flow cytometry. The method of claim 1, wherein the quantum dots are quantum dots emitting fluorescence at 525 nm and quantum dots emitting fluorescence at 655 nm. The quantum dot detection probe of claim 6 or 7, wherein the quantum dot detection probe is attached to the quantum dot emitting fluorescence at 525 nm, and the benzo [B] is applied to the quantum dot emitting fluorescence at 655 nm. A method for detecting contaminants in a sample using a quantum dot-based competitive immunoassay and multiple flow cytometry characterized in that the quantum dot detection probe attached to a pyrene antigen.

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