Portable nanoaptamer analyzer for detection of bisphenol A

Abstract

The present invention relates to a portable analyzer for detecting bisphenol A, which comprises an aptamer specifically binding to bisphenol A, and a method for detecting bisphenol A using the same. The analyzer of the present invention can analyze a small amount of a sample collected from a contaminated environment in a field at a level similar to a laboratory environment, thereby having an effect of enabling a more immediate and accurate detection and quantification of bisphenol A.

Claims

1. A portable analyzer for detecting bisphenol A, comprising: (a) a reaction unit, which comprises: (i) a first complex comprising a magnetic bead, a first labeling material, a second labeling material, and an aptamer specifically binding to bisphenol A; (ii) a reaction vessel having an inlet, wherein the first complex reacts with bisphenol A to form a second complex comprising the magnetic bead, the first labeling material, and the aptamer and the second labeling material is separated from the first complex; and (iii) a means for collecting the second complex in the reaction vessel having a magnetic force source for applying a magnetic force to the reaction vessel in an ON/OFF manner in order to collect the second complex; and (b) a detection unit having a means for detecting signals generated from the first and second labeling materials having a photodiode, a charge integrator, or a charge amplifier; and wherein the aptamer consists of the nucleotide sequence of SEQ ID NO: 1.

2. The analyzer according to claim 1, wherein the reaction vessel comprises the first complex.

3. The analyzer according to claim 1, wherein the reaction unit further comprises (iv) a means for removing the first complex and bisphenol A, which did not form a second complex, and the separated second labeling material, wherein the means for removing further comprises: a washing solution vessel comprising a washing solution; and a means for transporting the washing solution from the washing solution vessel to the reaction vessel to remove the first complex and bisphenol A, which did not form the second complex, and the separated second labeling material with the washing solution, wherein the means for transporting the washing solution has a miniature peristaltic pumps.

4. The analyzer according to claim 3, wherein the reaction unit further comprises (v) a vibrating element for vibrating the reaction vessel and preventing settling of the second complex.

5. The analyzer according to claim 1, wherein the detection unit further comprises a signal-generating means for generating signals from the first labeling material and the second labeling material, respectively, wherein the signal-generating means comprises a light-emitting diode (LED), a laser diode (LD), a vertical-cavity surface-emitting laser, a semiconductor diode, or a mercury lamp.

6. The analyzer according to claim 1, wherein each of the first labeling material and the second labeling material is a quantum dot, a fluorescent dye, a radiolabel, or an electrochemical functional group.

7. The analyzer according to claim 1, which further comprises a heat sink or a fan.

8. The analyzer according to claim 5, which further comprises a microcontroller board controlling the means for collecting the second complex, the signal-generating means, and the detection unit in an ON/OFF manner.

9. A method for detecting bisphenol A using the analyzer of claim 1, comprising injecting a collected sample into the inlet of the reaction vessel.

10. The method according to claim 9, wherein the injection of the sample into the inlet is performed by simultaneously or sequentially injecting a collected sample, and a first complex comprising a magnetic bead, a first labeling material, a second labeling material, and the aptamer.

11. A method for detecting bisphenol A using the analyzer of claim 2, comprising injecting a collected sample into the inlet of the reaction vessel.

12. A method for detecting bisphenol A using the analyzer of claim 3, comprising injecting a collected sample into the inlet of the reaction vessel.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1a is a diagram of a mechanism for detecting bisphenol A (BPA) in the portable analyzer of the present invention for detecting BPA. A complex containing an aptamer for detecting BPA is shown.

(2) FIG. 1b is a diagram illustrating the portable analyzer for detecting BPA, which does not include the control unit according to an embodiment of the present invention.

(3) FIG. 2 is a plan view of the portable analyzer according to an embodiment of the present invention for detecting BPA.

(4) FIG. 3 is a schematic diagram of the charge integrator according to an embodiment of the present invention.

(5) FIG. 4 is a diagram illustrating a PCB layout of the charge integrator according to an embodiment of the present invention.

(6) FIG. 5a is an operation schematic of the portable analyzer according to an embodiment of the present invention for detecting BPA.

(7) FIG. 5b is a drawing of the portable analyzer according to an embodiment of the present invention for detecting BPA.

(8) FIG. 6 is a diagram illustrating the control unit according to an embodiment of the present invention.

(9) FIGS. 7a-7e are graphs illustrating the results of setting a baseline in the analyzer according to an embodiment of the present invention for detecting BPA. FIG. 7a is a graph illustrating the normalized output signal (CH2/CH1) of five individual empty reaction vessels. FIG. 7b is a graph illustrating the normalized output signal for the reagent volume in the reaction vessel. FIG. 7c is a graph illustrating the normalized output signal for both empty reaction vessel and Tris-HCl buffer-filled reaction vessel (with and without vibration). FIG. 7d is a graph illustrating the voltage gradient of QD.sub.565 (CH1) and QD.sub.655 (CH2) serially diluted in a Tris-HCl buffer, which was measured by the analyzer according to the present invention for detecting BPA. FIG. 7e is a graph illustrating the fluorescence of samples in which QD.sub.565 and QD.sub.655 are serially diluted in a Tris-HCl buffer, which was measured by the commercial spectrofluorometer.

(10) FIGS. 8a-8e are graphs illustrating the results after incubation and rinsing processes in a laboratory environment, not in the portable analyzer according to the present invention for detecting BPA. FIG. 8a is a graph illustrating samples at 0, 5, 15, and 30 minutes without vibration (left) and with vibration (right). FIG. 8b is a graph illustrating the result of performing fluorescence measurement by the portable analyzer for detecting BPA without vibration. FIG. 8c is a graph illustrating the result of performing fluorescence measurement by the portable analyzer for detecting BPA with vibration. FIG. 8d is a graph illustrating the result of fluorescence measurement by the commercial spectrofluorometer. FIG. 8e is a graph illustrating fluorescence measurement correlation between the portable analyzer of the present invention for detecting BPA and the commercial spectrofluorometer.

(11) FIGS. 9a-9c are graphs illustrating the normalized fluorescence of BPA (F655/F565) at three different concentrations (0.5 ng/mL, 1.0 ng/mL, and 5.0 ng/mL) according to the incubation times (5, 15, 30, and 45 minutes). FIG. 9a shows the result without vibration. FIG. 9b shows the result with vibration. FIG. 9c is a graph illustrating the normalized fluorescence according to the rinse cycle (1.0 ng/mL).

(12) FIGS. 10a-10e are graphs illustrating the results after incubation and rinsing processes conducted in the portable analyzer according to the present invention for detecting BPA. FIG. 10a is a graph illustrating the result of fluorescence measurement by the portable analyzer for detecting BPA without vibration. FIG. 10b is a graph illustrating the result of fluorescence measurement by the portable analyzer for detecting BPA with vibration. FIG. 10c is a graph illustrating the result of fluorescence measurement by the commercial spectrofluorometer. FIG. 10d is a graph illustrating the correlation between incubation and rinsing processes of off-system and on-system. FIG. 10e is a graph illustrating the correlation between the portable analyzer according to the present invention for detecting BPA and laboratory protocol (off-system incubation+rinsing+commercial spectrofluorometer).

DETAILED DESCRIPTION OF THE INVENTION

(13) Hereinbelow, the present invention will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention.

Example 1: Design of Portable Analyzer for Detecting BPA

(14) The present inventors have tried to develop a portable analyzer for detecting BPA directly in a contaminated environment, and as a result, they have designed an analyzer including an aptamer capable of specifically detecting BPA, in which the reaction and analysis can be performed in a single reaction vessel (cuvette) (FIGS. 1 and 2).

(15) The detection of BPA using the portable analyzer of the present invention for detecting BPA can be carried out in three steps: (i) incubation, (ii) rinsing, and (iii) fluorescence measurement. The components of the analyzer of the present invention are as follows:

(16) The primary components of the analyzer consist of (i) a cuvette holder; (ii) miniature peristaltic pumps (Dolomite Miniature Peristaltic Pump, 3 V DC, 0.12 W, 0.45 mL/min, Dolomite Centre Ltd, Royston, UK) for transferring reagents; (iii) an articulated magnet (Neodymium, D42-N52, Disc , K&J Magnetics Inc, Pennsylvania, USA) mounted on a servo motor (TowerPro SG90 Servo, 4.8 V, Taiwan); (iv) a vibrating motor (Model Z7AL2B1690002, up to 12,000 rpm, Jinlong Machinery and Electronics Co. Ltd, China) for reagent agitation; (v) an LED array (10 W, 400 nm to 405 nm, Epiled, China) for excitation of quantum dots; (vi) a pair of photodiodes for detection (S6430-01 and S6429-01, Hamamatsu Photonics K.K., Japan); and (vii) a charge integrator to measure the output from the photodiodes. The LED array is further fitted with a heat sink and fan to prevent overheating.

(17) Additionally, the components may be controlled by an Arduino compatible Mega2560 microcontroller board via a 162 LCD and key pad (FIG. 5a).

(18) The charge integrator was custom-designed and manufactured. It was designed by including a low cost precision operational amplifier (LTC1051, Linear Technologies, USA), a 10 F feedback capacitor, and 2 k feedback resistor (FIGS. 3 and 4).

(19) The photodiodes in the charge integrator were used to convert signals, which are changed depending on whether BPA and the aptamer are bound, into electrical charges (electrons), and the converted electrical charges were accumulated in a feedback capacitor to generate a voltage drop of the feedback capacitor (or the voltage drop of the charge integrator output).

(20) The output of the charge integrator was 0 V to 5 V, and the outputted voltage was recorded using a data logger (PCS10, 4-Channel Recorder, Velleman, UK). The voltage outputs of the 540 nm and 660 nm photodiodes (via the charge integrator) were recorded by the data recorder as CH1 and CH2, respectively.

(21) The measured normalized output signal of the portable analyzer for detecting BPA was calculated as follows:

(22) $\mathrm{Normalized}\mathrm{output}\mathrm{signal}=\frac{\mathrm{Voltage}\mathrm{gradient}\mathrm{of}\mathrm{CH}2}{\mathrm{Voltage}\mathrm{gradient}\mathrm{of}\mathrm{CH}1}$

(23) The voltage gradient was calculated by dividing the change in output voltage from the charge integrator over the time duration.

(24) The LED array for quantum dot excitation was powered via a parallel 9 V line with a serial potentiometer. The potentiometer determined the final voltage of the LED array, and the 9 V line (a 5 V voltage regulator; LM7805A, Fairchild Semiconductor, USA) supplied power to both the fan and charge integrator. Electrical relays (TQ2-5V, Matsushita Electric Works, Japan) were used for switching the LED array as well as resetting the charge integrator.

(25) During operation of the analyzer of the present invention, the user controlled the peristaltic pumps, servo motor and articulated magnet, vibrating motor, and LCD array via the control unit (FIG. 6).

(26) A vibration motor was used to maintain homogeneity of the samples during the incubation as well as the fluorescence measurement. The agitation using the vibration motor can be easily facilitated by suspending the reaction vessel (cuvette) holder from a plastic pivot. During the fluorescence measurement, a cap was placed on the cuvette to minimize background light.

(27) The NanoAptamer analyzer according to the present invention, which has the above-described constitution, is a small-sized device, the size of which is similar to the size of the palm of a hand (FIG. 5b).

Example 2: Characterization of Baseline of Portable Analyzer for Detecting BPA

(28) Baseline characterization of the portable analyzer for detecting BPA was necessary to establish the operational limits of the analyzer prior to actual BPA detection and quantification. Accordingly, the present inventors performed the baseline characterization experiment in the following steps:

(29) (i) Response of empty cuvettes during fluorescence measurement

(30) (ii) Response with various reagent volumes (Tris-HCl buffer)

(31) (iii) Cuvette vibration during fluorescence measurement

(32) (iv) Fluorescence measurement of quantum dots only (QD.sub.565 and QD.sub.655)

(33) (v) Cuvette vibration to minimize settling of complexes

(34) (vi) Fluorescence measurement of MB-QD.sub.565 complex only

(35) The steps above will be described in more detail as follows:

Example 2-1: Response of Empty Cuvettes During Fluorescence Measurement

(36) In the Example, the present inventors used UV/visible range semi-micro cuvettes (Kartell, Millan, Italy). Since the cuvettes were made of polymethylmethacrylate (PMMA), individual cuvettes might have slight changes, which in turn cause variations during fluorescence measurement.

(37) Therefore, background optical characterization of empty cuvettes was required to be performed before using the analyzer of the present invention.

(38) To do this, five different empty cuvettes were inserted into the analyzer of the present invention, and the fluorescence measurement step (three times per cuvette) was performed. The fluorescence measurement step consisted of illumination by a UV-LED and measurement by photodiodes. The voltage outputs of 540 nm and 660 nm photodiodes (via the charge integrator) were recorded by a data recorder (PCS10, 4-Channel Recorder, Velleman, UK) as CH1 and CH2, respectively. The normalized output signals were calculated and compared.

Example 2-2: Response with Various Reagent Volumes

(39) Additionally, in order to determine the optimized reagent volume in the cuvette for fluorescence measurement for the detection of BPA, the present inventors prepared cuvettes filled with a Tris-HCl buffer (0.02 mol/L, pH 8, 0.005% SDS).

(40) The Tris-HCl buffer consisted of 0.02 mol/L of Tris-HCl (Sigma-Aldrich Co., St. Louis, USA), 0.02 mol/L of MgCl.sub.2.6H.sub.2O (Daejung, Gyung-gi, Korea), 0.04 mol/L of KCl (Duksan, Gyung-gi, Korea), and 0.1 mol/L of NaCl (Junsei, Tokyo, Japan). Additionally, sodium dodecyl sulfate (SDS, Sigma-Aldrich Co.) was added to the Tris-HCl buffer (0.02 mol/L, pH 8) so that the final concentration reached 0.005% (V/V). Cuvettes containing various volumes of the Tris-HCl buffer (0.02 mol/L, pH 8, 0.005% SDS) between 100 L and 1000 L were inserted into the analyzer of the present invention to perform fluorescence measurement and the normalized output signals were compared.

Example 2-3: Confirmation of Effect of Cuvette Vibration During Fluorescence Measurement

(41) In order to confirm the background effect of cuvette vibration during fluorescence measurement by the analyzer of the present invention, an empty cuvette and a Tris-HCl buffer (1000 L)-filled cuvette were compared. Fluorescence measurement was performed with and without vibration (in triplicate), and the normalized output signals obtained were compared.

Example 2-4: Fluorescence Measurement of Quantum Dots Only

(42) In order to confirm the fluorescence measurement range of the analyzer of the present invention, the present inventors used serially diluted quantum dots in the Tris-HCl buffer.

(43) Specifically, commercial carboxyl quantum dots QD.sub.565 and QD.sub.655 (Invitrogen, Carlsbad, USA) were procured as an 8 M stock. The test samples used in the Example had a volume of 1000 L (in cuvettes) with a final QD concentration of 10.sup.1 mol/L to 10.sup.6 mol/L.

(44) The test samples were subjected to fluorescence measurement by both the analyzer of the present invention and a commercial spectrofluorometer (Molecular devices, SpectroMax M2 microplate reader, Sunnyvale, USA). The excitation and emission wavelengths of the quantum dots were 360 nm and 570 nm for QD.sub.565, and 360 nm and 660 nm for QD.sub.655, respectively.

Example 2-5: Confirmation of Effect of Cuvette Vibration to Minimize Settling of Complexes

(45) In order to minimize settling due to the weight of magnetic beads of the complexes during incubation or fluorescence measurement, a vibrating element capable of vibrating cuvettes was included in the analyzer of the present invention.

(46) The samples (200 L) were each incubated for 0 minutes, 5 minutes, 15 minutes, and 30 minutes with and without vibration caused by the vibrating element. Post-incubation photographs were taken to compare the degree of settling.

Example 2-6: Fluorescence Measurement of MB-QD.SUB.565 .Complex Only

(47) Since magnetic beads were significantly larger and heavier than quantum dots, these may interfere fluorescence measurement of the analyzer of the present invention. Therefore, the present inventors examined fluorescence measurement in the presence of magnetic beads.

(48) Specifically, the aminated magnetic beads (MB, Invitrogen) were covalently bonded with carboxylated QD.sub.565 to form an MB-QD.sub.565 complex. The complex was diluted with the Tris-HCl buffer to a final volume of 1000 L. Fluorescence measurement by the analyzer of the present invention was performed on three test samples with the same complex in triplicate with and without vibration.

Example 3: Detection and Quantification of BPA by Portable Analyzer for Detecting BPA

(49) The capability of the analyzer of the present invention to detect and quantify BPA was demonstrated through the following steps. (i) On-system fluorescence measurement of MB-QD-BPA complex (off-system incubation and rinsing) (ii) Optimization of on-system incubation duration (iii) Optimization of on-system rinsing cycle (iv) On-system incubation, rinsing, and fluorescence measurement

(50) The steps above will be described in more detail as follows:

Example 3-1: Fluorescence Measurement of MB-QD-BPA Complex (Off-System Incubation and Rinsing)

(51) The MB-QD.sub.565-aptamer-QD.sub.655 complex was prepared using the BPA-specific aptamer (24 bp, NH.sub.2-C.sub.6-T.sub.10-GGATAGCGGGTTCC, SEQ ID NO: 1).

(52) Specifically, the signaling probe (30 bp, NH.sub.2-C.sub.6-T.sub.10-TATCCCACCTGACCACCCAC, SEQ ID NO: 2) labeled with QD.sub.655 was hybridized with the MB-QD.sub.565 complex prior to incubation with BPA.

(53) BPA was dissolved in methanol (Duksan, Korea) as a stock solution. Thereafter, the BPA stock solution was serially diluted in deionized water to achieve various concentrations of 0 ng/mL to 1.0 ng/mL (ppb).

(54) The prepared MB-QD.sub.565-aptamer-QD.sub.655 complex was incubated with BPA with the concentrations of 0 ng/mL, 0.0005 ng/mL, 0.001 ng/mL, 0.01 ng/mL, 0.1 ng/mL, and 1.0 ng/mL (ppb) on a bench shaker (Eppendorf, MixMate, Hamburg, Germany) for 2 hours to utlimately form a MB-QD-BPA complex.

(55) After incubation, the formed MB-QD-BPA complex was manually separated using a magnet (Invitrogen, DynaMag-2) and rinsed with the Tris-HCl buffer to form samples (test samples) with a volume of 200 L.

(56) The test samples prepared in the above were measured by the commercial spectrofluorometer (SpectraMax M2 microplate reader, Molecular Devices, CA, USA). This would also verify the successful completion of the reaction.

(57) Further, for fluorescence measurement by the analyzer of the present invention, the test samples were diluted 5-fold with the Tris-HCl buffer to prepare samples having a final volume of 1000 L.

Example 3-2: Optimization of On-System Incubation Duration

(58) In order to optimize the duration for on-system incubation by the analyzer of the present invention, test samples with three different concentrations of BPA (i.e., 0.5 ng/mL, 1.0 ng/mL, and 5.0 ng/mL, or ppb) were prepared. At each concentration, the test samples were incubated for 5 minutes, 15 minutes, 30 minutes, and 45 minutes with and without vibration.

(59) After incubation, the test samples were subjected to fluorescence measurement via the commercial spectrofluorometer (SpectraMax M2 microplate reader) at the wavelengths of 360 nm for excitation and 570 nm (QD.sub.565) or 660 nm (QD.sub.655) for emission. The results were presented as normalized fluorescence, which is given by the following equation:

(60) $\mathrm{Normalized}\mathrm{fluorescence}=\frac{\mathrm{Fluorescence}\mathrm{of}\mathrm{QD}655}{\mathrm{Fluorescence}\mathrm{of}\mathrm{QD}565}$

Example 3-3: Optimization of On-System Rinsing Cycle

(61) Four samples with the same amount of BPA (1.0 ng/mL or ppb) were prepared and incubated on-system for 30 minutes.

(62) After on-system incubation, on-system rinsing was performed via miniature peristaltic pumps and an articulated magnet.

(63) Specifically, the cuvette was rinsed with the Tris-HCl buffer using peristaltic pumps while the magnet was deployed to the near-surface of the cuvette to collect the MB-QD-BPA complexes. After each rinse, the complexes were re-suspended via vibrating the cuvette. Single and double rinse cycles were performed and the resulting normalized fluorescence was measured and compared.

Example 3-4: On-System Incubation, Rinsing, and Fluorescence Measurement

(64) In this Example, the capability of the analyzer of the present invention to perform on-system incubation, rinsing, and fluorescence measurement was demonstrated.

(65) First, the MB-QD complex was prepared in the same manner as in the Example above. The prepared complex was incubated with BPA of 0 ng/mL, 0.0005 ng/mL, 0.001 ng/mL, 0.01 ng/mL, 0.1 ng/mL, and 1.0 ng/mL (ppb) for 30 minutes in the reaction vessel of the analyzer of the present invention by exerting vibration.

(66) After incubation, the articulated magnet and miniature peristaltic pumps were operated to transport the Tris-HCl buffer to the reaction vessel in which the reaction had been carried out, and then a double rinse cycle was performed.

(67) After rinsing, the complex was diluted 5-fold to a final volume of 1000 L prior to fluorescence measurement. The normalized output signal by the analyzer of the present invention was compared with that by the commercial spectrofluorometer.

(68) The results of the experiments according to the Example above were analyzed as in the following Experimental Examples.

Experimental Example 1: Baseline Characterization of Portable Analyzer for Detecting BPA

Experimental Example 1-1: Response of Empty Cuvettes During Fluorescence Measurement

(69) The average normalized output signal and standard deviation of five individual empty cuvettes ranged from 0.5561 to 0.5986 and 0.0007 to 0.0025, respectively (FIG. 7a). That is, more variation was observed between individual empty cuvettes than repeated fluorescence measurement by the analyzer of the present invention.

Experimental Example 1-2: Response Result with Various Reagent Volumes

(70) It was confirmed that when the reagent volume was less than 600 L, the normalized output signal obtained as a result of fluorescence measurement fluctuated (FIG. 7b).

(71) However, it was confirmed that when the reagent volume was increased beyond 600 L, the output signal results had a relatively constant value (0.5628+0.0037).

(72) Therefore, in order to obtain consistent fluorescence measurement results, fluorescence measurement of the analyzer of the present invention was performed using the reagent volume (1000 L) in the following Examples. Since the reagent volume in which the reaction occurred in the reaction vessel of the analyzer was 200 L, the reagent volume was diluted 5-fold, prior to fluorescence measurement by the analyzer of the present invention.

Experimental Example 1-3: Confirmation of Influence of Cuvette Vibration During Fluorescence Measurement

(73) The present inventors tried to confirm influence of the presence of vibration of cuvettes during fluorescence measurement. As a result, empty cuvettes showed normalized output signals at 0.5510 and 0.5776 for vibration-off and vibration-on, respectively (FIG. 7c and Table 1).

(74) TABLE-US-00001 TABLE 1 Normalized output signals of empty cuvettes and Tris-HCl buffer-containing cuvettes CH2/CH1 MEAN STD Empty cuvette (without vibration) 0.5510 0.0033 Empty cuvette (with vibration) 0.5776 0.0018 Tris-HCl buffer (without vibration) 0.5460 0.0030 Tris-HCl buffer (with vibration) 0.5561 0.0011

(75) For the cuvettes filled with the Tris-HCl buffer, the results were similar at 0.5460 and 0.5561. As expected, the standard deviation for the empty cuvette with vibration (at 0.0018) was lower than that without vibration (at 0.0033).

(76) A similar trend was observed with the Tris-HCl buffer-filled cuvettes. Specifically, it was confirmed that the standard deviation for the output signal of the Tris-HCl buffer-filled cuvette with vibration was at 0.0011, which was lower than that for the output signal of the Tris-HCl buffer-filled cuvette without vibration (at 0.0030).

(77) It was confirmed from the results above that when vibration was applied to the reaction vessel during the fluorescence measurement process for detecting BPA, the standard deviation of the measured output signals was reduced.

Experimental Example 1-4: Fluorescence Measurement of Quantum Dots Only

(78) Concentrations of QD.sub.565 and QD.sub.655 were measured with the portable analyzer of the present invention for detecting BPA, and the results were confirmed as voltage gradients.

(79) As a result, it was confirmed that the concentrations of the quantum dots QD.sub.565 and QD.sub.655 were from 10.sup.3 mol/L to 10.sup.1 mol/L and 10.sup.5 mol/L to 10.sup.1 mol/L, respectively (FIG. 7d).

(80) At each concentration within the measurement range, QD.sub.655 exhibited a higher voltage gradient (fluorescence) than QD.sub.565. The linear regression curve for QD.sub.565 was given as log.sub.10(y)=0.55 log.sub.10(x)+0.59 (r.sup.2=0.96) for a concentration range of 10.sup.3 mol/L to 10.sup.1 mol/L. Further, the linear regression curve for QD.sub.655 was given as log.sub.10(y)=0.64 log.sub.10(x)+1.68 (r.sup.2=0.95) for a concentration range of 10.sup.5 mol/L to 10.sup.1 mol/L.

(81) As expected by the present inventors, fluorescence measurement of the same samples by a commercial spectrofluorometer yielded steeper linear regression slopes compared to the analyzer of the present invention (FIG. 7e). The linear regression curve for QD.sub.565 was given as log.sub.10(y)=1.0 log.sub.10(x)+5.33 (r.sup.2=0.999) for a concentration range of 10.sup.4 mol/L to 10.sup.1 mol/L. The linear regression curve for QD.sub.655 was given as log.sub.10(y)=1.03 log.sub.10(x)+5.99 (r.sup.2=0.997) for a concentration range of 10.sup.5 mol/L to 10.sup.1 mol/L.

Experimental Example 1-5: Cuvette Vibration to Minimize Settling of Complexes

(82) After conducting incubations with and without vibration, the degree of settling of the samples was compared (FIG. 8a).

(83) Using 0 minutes as a negative control, no significant difference was observed in the settling of the samples during the incubation time (5 minutes). However, for the sample incubated for 15 minutes, settling of the complexes became apparent in the absence of vibration. In addition, after 30 minutes, it was confirmed that the settling was completed and that the supernatant liquid became transparent.

(84) From these results, the present inventors confirmed that when BPA was detected using the analyzer of the present invention, the incubation and fluorescence measurement can be facilitated by vibrating the reaction vessel, and as a result, the efficiency of the analyzer for detecting BPA could be increased.

Experimental Example 1-6: Fluorescence Measurement of MB-QD.SUB.565 .Complex Only

(85) The three test samples including the MB-QD.sub.565 complex exhibited the normalized output signals that are similar to each other (Table 2). The total average values that corresponded to with and without vibration are 0.4610 and 0.4527. With vibration, the standard deviation ranged from 0.0016 to 0.0042. Such standard deviation was smaller than that without vibration, where it ranged from 0.0034 to 0.0123. Fluorescence measurement with vibration showed a reduced standard deviation compared to that without vibration.

(86) Such results seemed to be exhibited because the vibration applied to the reaction vessel prevented the aggregation and settling of the MB-QD.sub.565 complex in the samples, thereby allowing more consistent fluorescence measurement.

(87) TABLE-US-00002 TABLE 2 The normalized output signals (CH2/CH1) of the MB-QD.sub.565 complex, which are measured through the portable analyzer for detecting BPA without and with vibration CH2/CH1 1 2 3 Average std Without vibration #1 0.4652 0.4598 0.4546 0.4599 0.0053 #2 0.4637 0.4597 0.4570 0.4601 0.0034 #3 0.4504 0.4382 0.4258 0.4382 0.0123 Average 0.4527 0.0129 With vibration #1 0.4665 0.4721 0.4730 0.4705 0.0035 #2 0.4611 0.4652 0.4695 0.4653 0.0042 #3 0.4491 0.4461 0.4465 0.4472 0.0016 Average 0.4610 0.0110

Experimental Example 2: Detection and Quantification of BPA Using Portable Analyzer for Detecting BPA

Experimental Example 2-1: Fluorescence Measurement of MB-QD-BPA Complex (Off-System Incubation and Rinsing)

(88) BPA (0.0005 ng/mL to 1 ng/mL (ppb)) of the samples, which were subjected to off-system incubation and rinsing was detected and quantified by using the portable analyzer of the present invention (FIGS. 8b and 8c). The linear regression curve of the resulting normalized output signal was given as y=0.24 log.sub.10(x)+5.40, r.sup.2=0.91, when vibration was not present. The linear regression curve thereof was given as y=0.15 log.sub.10(x)+5.76, r.sup.2=0.75, when vibration was present.

(89) Similar to the results of the Examples above, the fluorescence measurement values in the presence of vibration showed significantly smaller standard deviation compared to those without vibration. The lower r.sup.2 in the presence of vibration was attributable to the measurement at 0.0005 ng/mL (ppb).

(90) The results of measuring fluorescence by the analyzer of the present invention were consistent with the results measured by a commercial spectrometer. The linear regression curve measured with a commercial spectrofluorometer was 1.42 log.sub.10(x)+16.08 with r.sup.2=0.98.

(91) The fluorescence measurement correlation between the analyzer of the present invention and the commercial spectrofluorometer was confirmed using samples which were subjected to the same off-system (laboratory) incubation and rinsing processes (FIG. 8e). The correlation coefficient r was 0.91.

(92) From the results above, the present inventors confirmed that the results obtained with the analyzer of the present invention were the same as the results of measuring fluorescence by the commercial spectrofluorometer, and that the analyzer of the present invention was able to detect BPA at an equivalent level compared to that detected in a laboratory environment.

Experimental Example 2-2: Optimization of On-System Incubation Duration

(93) For incubation with vibration, a minimum of 30 minutes was required for sufficient incubation (red dotted box in FIG. 9b). As the BPA concentration increased, the normalized fluorescence was reduced.

(94) However, in the absence of vibration, the trend according to the reaction was not observed even after 45 minutes (FIG. 9a).

(95) From the results above, it was confirmed that when BPA was detected using the analyzer of the present invention, successful BPA detection was able to be performed due to the prevention of the settling of the complexes by exerting vibration.

Experimental Example 2-3: Optimization of On-System Rinsing Cycle

(96) The present inventors tried to confirm a rinse cycle optimized for the detection of BPA using the analyzer of the present invention.

(97) As a result, it was confirmed that the normalized fluorescence by a double rinse cycle showed more consistent results as compared to that by a single rinse cycle (FIG. 9c). In particular, samples #2, #3, and #4 showed similar results at 2.771 (+0.877) after the double rinse cycle.

(98) Therefore, the double rinse cycle was used for subsequent on-system rinsing.

Experimental Example 2-4: On-System Incubation, Rinsing, and Fluorescence Measurement

(99) As a result of measuring on-system incubation, rinsing, and fluorescence using the analyzer of the present invention, it was confirmed that the samples showed a reaction similar to that when the samples were subjected to off-system incubation and rinsing (FIGS. 10a and 10b).

(100) In the absence of vibration, the linear regression curve was given as y=0.20 log.sub.10(x)+2.32, r.sup.2=0.89; and in the presence of vibration, the linear regression curve was given as y=0.21 log.sub.10(x)+2.81, r.sup.2=0.90.

(101) As expected, the standard deviation of fluorescence measurement in the presence of vibration was significantly smaller than that in the absence of vibration. FIG. 10c shows the normalized fluorescence of the same on-system incubated and rinsed samples measured by the commercial spectrofluorometer. Such fluorescence exhibited a linear regression curve given as y=0.27 log.sub.10(x)+6.30, r.sup.2=0.89.

(102) In order to establish equivalence between on-system and off-system incubation as well as rinsing, the correlation was investigated (FIG. 10d). In both cases, fluorescence measurement was performed using the commercial spectrometer. As a result, for the BPA concentration ranging from 0.0005 ng/mL to 1 ng/mL (ppb), the correlation coefficient for both off-system and on-system incubation and rinsing was r=0.92.

(103) Finally, the present inventors compared the results of the BPA detection and quantification measured by using the laboratory protocol (off-system incubation+rinsing+commercial spectrometer) with those measured by using the analyzer of the present invention (on-system incubation+fluorescence measurement via LED array/photo diodes). As a result, it was confirmed that the correlation coefficient was r=0.72 (FIG. 10e).

(104) From the results of the Examples above, it was confirmed that the results of incubating and rinsing the samples in the analyzer of the present invention were similar to those of performing the same with the laboratory protocol.

(105) It was also confirmed that BPA was able to be sufficiently detected by incubating the collected samples and measuring fluorescence by the miniaturized analyzer of the present invention.

(106) That is, the results indicate that the analyzer of the present invention can replace the existing laboratory protocol, and that a system capable of detecting and quantifying BPA at environmentally relevant concentrations (<1 ng/mL or ppb) can also be implemented even with a miniaturized device.

(107) From the foregoing, one of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.