JP7012301B2 - Drug detection system - Google Patents

Description

The present invention relates to a drug detection system, and more particularly to a drug detection system that detects a drug to be detected by using electrochemical luminescence to distinguish it from other substances.

Abuse of illicit drugs that are highly addictive and harmful to human health has become a serious global challenge, with amphetamines being a powerful central nervous system stimulant that induces sleeplessness and loss of appetite. It is recognized as a stimulant) and is subject to crackdown. And since most of the stimulants distributed in Japan are mainly composed of methamphetamine (hereinafter referred to as "MA"), MA contained in biological samples such as urine, blood and saliva and amphetamine which is a metabolite thereof can be accurately used. Detection has become an important part of the crackdown.

Many methods have been proposed for the detection of MA, and typical ones are instruments such as gas chromatography, liquid chromatography, capillary electrophoresis chromatography, and fluorescence photometric analysis combined with mass spectrometry. There is a detection method using an analyzer. Although these are detection means with high selectivity and sensitivity, they are applicable to analysis in the field of illegal drug detection due to the time and cost required for pretreatment and analysis and the large scale of measuring instruments. Can not.

On the other hand, as a method of identifying MA in the field such as detection, an immunoassay using a kit for detecting urinary abuse drugs such as Triage (registered trademark right holder: Aria San Diego Incorporated) (immunoassay analysis method) However, there is a problem that these test kits are relatively expensive. Further, as a method for detecting MA at a lower cost, a method using discoloration due to a chemical reaction with a Marquis reagent / Simon reagent is used, but it has a drawback of relatively low selectivity. Therefore, it has been required to develop a method having a different detection principle for detecting MA more accurately.

Therefore, the electrochemical luminescence (hereinafter referred to as "ECL") method is being studied as a drug detection method in biological samples that is expected to be applied in the field and highly selective.
ECL is a phenomenon in which a luminescent chemical species generated by an electrode reaction is excited by a subsequent chemical reaction and emits light (Non-Patent Documents 1 and 2). By using this as a drug detection means, the following features are obtained. Is said to be obtained. (Non-Patent Document 2-4).
-Because an excitation light source such as fluorescence spectroscopy is not required, the background signal from the light source becomes small.
-Since luminescence is triggered by an electrode reaction, the luminescence location and luminescence time can be easily controlled as compared with chemiluminescence.
-Since the equipment is small, it is possible to perform on-site analysis at various sites.
-Even translucent biological samples such as urine and blood can be analyzed by devising the detector configuration.

Under such circumstances, the present inventors have proposed a method for detecting a drug by ECL using a potential sweep method in which the ECL intensity is measured while linearly sweeping the electrode potential. In this document, the β2 receptor stimulant is a drug that is similar in structure to MA (see FIG. 2) and that makes it difficult to identify MA by showing a false positive with the above-mentioned Simon reagent. The future potential for distinction from methoxyphenamine (hereinafter referred to as MP) (see FIG. 3), which is classified as a bronchodilator and legally prescribed as a bronchodilator, was shown. (Non-cited document 5)

Electrogenerated Chemiluminescence; Dekker: New York, US, 2004. Miao, W. Chemical Reviews 2008, 108, 2506-2553. Jin, J .; Muroga, M .; Takahashi, F .; Nakamura, T. Bioelectrochemistry 2010, 79, 147-151. Takahashi, F .; Jin, J. Analytical and Bioanalytical Chemistry 2009, 393, 1669-1675. Fumiki, T .; Saki, N .; Hirosuke, T .; Jiye, J .; Teruo, H. In 54th Annual meeting of the International Association of Forensic Toxicologists: Brisbane, Australia, 2016, p249

As described above, drug detection by ECL measurement can be adapted to drug detection in the field because the drug in the biological sample can be screened with high selectivity depending on the detected drug, and it can also be stored in a relatively small system. It shows the possibility.

Further, according to Non-cited Document 5, Ru (bpy) 32+ (tris (2,2-bipyridine) ruthenium (ll)), which is generally used as an ECL probe, is used as a luminescent species, and ECL measurement is performed using a potential sweep method. As a result, MA in the biological sample has only one emission peak observed at a specific voltage (1.2V), while the above-mentioned MP has a peak at the same voltage (1.2V) as MA and more. It was found that two peaks were observed at high voltage (1.45V), and by taking advantage of this difference in ECL profile, the ECL measurement showed the potential for selective screening of MA. There is. However, at the same time, with this method, it is difficult to clearly distinguish the difference in ECL profile between MA and MP due to the low absolute ELC intensity, especially when the MA concentration in the biological sample is at a low level. It has also been shown that it is difficult to distinguish, and if the biological sample contains a detection-interfering substance with similar properties such as MP in MA detection, it has an adverse effect on the detection of the target drug. The problem was left.

Therefore, the present invention reduces background noise during drug detection using ECL, and amplifies the observed ECL signal to increase the ECL intensity, thereby selectively screening the drug for drug detection. The challenge was to provide a system.

In addition, even if there is a drug with a similar ECL profile due to structural similarity, or even if the drug has a low concentration in the sample and is difficult to detect, erroneous detection can be prevented by changing the measured ECL profile. The challenge was to provide a drug detection system that can identify the target detection drug.

The drug detection system according to the present invention is a means for applying a continuous sine wave having a frequency in the range of 75 to 150 Hz as a sweep potential to an electrode in contact with the sample, and an emission signal obtained from the sample to which the sweep potential is applied. A means for simultaneously acquiring the AC cycle of the sweep potential, a means for performing arithmetic processing using the acquired emission signal and the AC cycle, and extracting an AC component of the emission signal, and the AC component. Is provided with a means for detecting the methanefetamine by comparing with the pattern of the methanefetamine .

The sweep potential that is periodically modulated as a continuous sine wave used in the drug detection system according to the present invention is generated by superimposing the AC potential of the continuous sine wave on the DC potential to be swept, for example, when the electrode is subjected to potential sweep. Is possible. As a method of generating a sweep potential that is periodically modulated as a continuous sine wave, a method of connecting a potentiometer that outputs a DC potential and a function generator that outputs an AC potential in series can be exemplified. Further, the potentiometer may be configured by an electrochemical analyzer in which the functions of the function generator are integrated.

As a method for extracting the AC component of the luminescence signal used in the drug detection system according to the present invention, for example, using a lock-in amplifier, the AC component of the sweep potential acquired at the same time as the luminescence signal is used as a reference frequency. The method of extracting is exemplified. At this time, if the noise component is removed by the low-pass filter built in the lock-in amplifier, the detection accuracy can be improved, which is preferable.

In the drug detection system according to the present invention, methamphetamine or a metabolite thereof can be exemplified as the drug to be detected, and in this case, a sample selected from urine, saliva, and blood can be exemplified as the sample used for detection. Is.

(Action)
It is suggested that the emission intensity of ECL is related to the rate at which the chemical reaction related to the emission occurs, and the sweep rate (the rate at which the potential applied to the electrode changes) and the ECL intensity change depending on the chemical species of interest. do. Then, by superimposing the AC potential on the DC potential of the present means, a rapid AC potential change occurs during the potential sweep, so that the apparent sweep speed can be temporarily increased. That is, changes in the ECL profile are observed, such as the ECL reaction cannot follow the potential change of the AC wave due to the difference in the rate at which the chemical reaction that causes luminescence occurs depending on the detected drug, and the luminescence reaction does not occur at the DC voltage that should occur. can.

A function generated by a function generator can be connected in series with a potentiostat, or an electrochemical analyzer having that function can output a signal obtained by superimposing a desired direct current and alternating current.

By removing noise with the low-pass filter of the lock-in amplifier and simultaneously capturing the measured data of the superimposed AC cycle and ECL signal at the same time, the lock-in amplifier automatically takes one cycle of the AC cycle from the synchronized signal. The difference between the maximum value and the minimum value can be calculated, and the difference between the maximum value and the minimum value in the one cycle can be integrated for several cycles or time with a lock-in amplifier, moved and averaged, and output as an analog signal. can.
Alternatively, it can be realized by connecting an electrochemical analyzer having that function to a personal computer and creating an arithmetic program.

Since the signal corresponding to the observed AC component of the ECL can be extracted and detected by signal processing by the lock-in amplifier or the calculation on the personal computer, the noise component in the ECL response by this measurement method can be dramatically reduced. ..

In the drug detection system according to the present invention, when the drug to be detected is MA, the frequency of the continuous sine wave is preferably 75 to 150 Hz, and more preferably 100 Hz. If the frequency is set too low, the ECL of the MP may be observed relatively strongly at the voltage at which the ECL intensity of the MA to be detected peaks, and it may be difficult to distinguish the frequency. If it is too high, the ECL intensity of both MA and MP tends to gradually decrease at a voltage at which the ECL intensity of MA and MP peaks, which may interfere with detection.

In the drug detection system according to the present invention, when the drug to be detected is MA, the amplitude of the continuous sine wave is preferably 20 to 100 mV, and more preferably 50 mV. In the analysis according to the present invention, when the amplitude is in the range of 10 mV to 200 mV, the ECL intensity of MA tends to increase according to the magnitude of the amplitude, and when the amplitude is too small, the ECL intensity becomes small and the analysis is difficult. Become. On the contrary, if the amplitude is set too large, the range of the potential of the measured ECL becomes large, so that it overlaps with the peak of the ECL of the MP, and there is a concern that it becomes difficult to distinguish.

According to the means and actions of the present invention, by reducing the baggage noise during drug detection by ECL, the measured emission signal can be amplified, and as a result, the relative intensity of ECL intensity is increased. This allows the detection drug to be selectively screened.

In addition, even if the ECL profile is close and difficult to identify due to similar structures such as MA and MP, or even if the concentration in the sample is low, the AC cycle that is superimposed during the potential sweep By appropriately setting the frequency and the like, the ECL profile can be changed, and as a result, identification can be performed.
In this way, by enabling clear identification, a drug detection system using ECL that can screen a specific drug in a single measurement without using a device such as a large-scale chromatograph for the target detection drug. Can be provided.

The schematic diagram which shows the apparatus system for the ECL measurement of embodiment of this invention. The chart which shows the result of the ECL measurement using the conventional potential sweep. A chemical reaction equation showing the emission scheme of MA. A chemical reaction formula showing the luminescence scheme of MP. The chart which shows the result of the ECL measurement using the potential sweep of the embodiment of this invention. The graph which shows the correlation of the frequency of alternating current and ECL intensity superposed in the said form. The graph which shows the correlation of the amplitude of the alternating current superposed in the said form, and the ECL intensity. The graph which shows the correlation between the MA concentration and the ECL intensity of the said form.

As an embodiment of the drug detection system using the ECL of the present invention, the detection test using the above-mentioned MA and MP as an example will be described in detail.

Before explaining this embodiment, the conventional MA and MP potential sweep ECL will be described. FIG. 2 shows the measurement results by the conventional potential sweep ECL, in which the upper row shows the potential profile 21 swept to the electrode, and the middle row shows the results of ECL emission (vertical axis) with respect to the MA sweep potential (horizontal axis) 22. , The lower part shows the same graph 23 in MP. The measurement conditions are the same as those measured by the system of the embodiment of the present invention described later, and there is a 10 μM MA (when the middle stage (a) of FIG. 2) or MP (when the lower stage (b) of FIG. 2). A detection solution containing Ru (bpy) 32+, which is a luminescent species of 100 μM, is used in a phosphate buffer solution (PBS; pH 9.0), and a DC potential sweep 20 mV / s; AC frequency 100 Hz; AC amplitude 50 mV; PMT voltage. It was measured as 500V.
3 and 4 show the chemical reaction between MA and MP and Ru (bpy) 32+, which is a luminescent species, at the time of ECL. FIG. 3 shows the reaction formula of MA and FIG. 4 shows the reaction formula of MP.

The potential profile 21 (upper part of FIG. 2) of the conventional potential sweep continuously rises with time by sweeping only by direct current. The result of ECL measurement is about 600a. At 1.2V in ECL measurement of MA. u. Only one peak was observed. On the other hand, the ECL measurement of MP is 1.2V and 1.45V, and two 500a. u. A peak of degree was observed, both of which were 50a. u. A degree of background noise 24 has been observed.

In the ECL measurement results, ECL emission was observed from 1.05 V, and the peak was observed at 1.2 V, which is almost the same as the oxidation potential from Ru (bpy) 32+ to Ru (bpy) 33+. It is considered that this is because MA and MP have a similar secondary amine skeleton and have a relatively stable intermediate radical that is electron-generated from the amine group by deprotonation. (See reaction equations (1) and (5) in FIGS. 3 and 4)

* Ru (bpy) 32+, which is a luminescent species, is generated by an electron exchange reaction between electrochemically generated Ru (bpy) 33+ (see reaction formula (2)) and the intermediate radicals of MA and MP. (See reaction equations (3) and (6)), and emission is observed when * Ru (bpy) 32+ returns to Ru (bpy) 32+. (See reaction equation (4))

On the other hand, in MP, the second peak is observed at 1.45V. This is because the benzyl cation radical is generated by electrochemical oxidation at a relatively high potential and is stabilized by the electron contribution effect of the methoxy group on the benzene ring. As a result, the intermediate radicals due to the electrochemical oxidation of MP last longer than MA. (See reaction equations (7) and (8))

From the above results, it is shown that MA and MP can be distinguished by potential sweep ECL measurement due to the difference in peak at 1.45V, but especially when the MP concentration in the sample solution is low, 1.2V. It could not be completely separated from the ECL signal, and it was difficult to measure with high sensitivity derived from the ECL behavior of 1.45V.

Next, the test of the potential sweep ECL measurement by superimposing the alternating current of MA and MP by the system of this embodiment will be described.
The biological sample used for this measurement is healthy human urine supplemented with MA standard solution, and the probe used as the luminescent species is Ru (bpy) 32+, which is generally used for ECL measurement. rice field.

FIG. 1 is a schematic diagram of a test device for ECL measurement by the system of this embodiment.
In this test device, a function generator 11 for superimposing an AC signal and a potentiostat 12 for outputting a direct current are connected in series, and a minute sinusoidal wave of the AC signal is superimposed on the direct current and modulated while the potential is swept. The applied potential is applied to the electrodes in the ECL cell 13. As for the voltage supply to the electrodes, any system such as an electrochemical analyzer that integrates the functions of the function generator 11 and the potentiometer 12 can be used as long as the potential modulation as described above is possible during the potential sweep. You may use it.

The ECL is measured in a microelectrolytic glass cell in the dark box 14, and a general three-electrode method using a glassy carbon working electrode, a silver / silver chloride reference electrode, and a platinum counter electrode is used. ECL, which is weak light emission from the electrode surface, was detected by a photoelectron doubling tube 15 equipped with a signal amplifier unit installed so as to face each other at a distance of 1.0 mm from the working electrode.

Further, the lock-in amplifier 16 is connected to the function generator 11 and the photomultiplier tube 15, and the sine wave of the superimposed periodic AC voltage applied to the electrodes and the observed ECL emission signal are synchronized and simultaneously. I took it in. Then, the observed light emission signal is amplified by the preamplifier 17 and the lock-in amplifier 16, and is digitized by entering the A / D converter 18 together with the current signal from the potentialostat 12 observed at the same time, and is output to the computer 19. And said. The lock-in amplifier 16 also functions as a low-pass filter for removing bag ground noise.

The detection solution in this test was adjusted as follows.
5.0 mL of urine containing MA hydrochloride (manufactured by Sumitomo Dainippon Pharma) was dispensed into a test tube, and 0.5 mL of 0.1 moldm-3 sodium hydroxide aqueous solution was added to make it alkaline.

1.0 mL of ethyl acetate was added to this solution, and the mixture was stirred with a shaker for 10 minutes. Then, using a 2000 x g centrifuge, centrifugation and separation were carried out for 10 minutes, and urine and ethyl acetate were phase-separated to extract MA in urine data to ethyl acetate.

0.10 mL of ethyl acetate after extraction was taken, and the pH was adjusted with phosphate buffered solution (PBS). 1.0 mmol dm-3 Ru (bpy) 32 + aqueous solution 0.50 mL, methanol 0.30 mL , To prepare a mixed solvent.

FIG. 5 shows the measurement results of the potential sweep ECL in which alternating current is superimposed and potential-modulated according to the present embodiment. The upper row shows the potential profile 51 that sweeps to the electrode, the middle row shows the ECL emission with respect to the sweep potential of MA, and the lower row Shows a similar graph 53 in MP. The measurement conditions were 100 μM Ru in a phosphate buffer solution (PBS; pH 9.0) in which 10 μM MA (in the middle (a) of FIG. 5) and MP (in the lower (b) of FIG. 5) were present, respectively. A detection solution containing (bpy) 32+ was used and measured at a DC potential sweep rate of 20 mV / s; AC frequency 100 Hz; AC amplitude 50 mV; PMT voltage 500 V.

In the potential profile 51 (upper part of the figure) of this example, a minute alternating current is superimposed on the direct current and the potential profile 51 rises with a periodic sine wave. As a result of the ECL measurement, in the ECL measurement of MA, the only peak of about 4500 a.u. Is observed at 1.15 V, which is a lower voltage than the conventional one. In MP, the previously observed peak of 1.2V is almost erased and becomes a weak peak, and a strong peak of about 3000a.u. Is observed at 1.45V. The relative background noise 54 is reduced as a whole, and the runout width is also small.

In this way, in the conventional ECL measurement, the peak at 1.2V that overlaps with MA is eliminated from the ECL profile of MP having two peaks, and the absolute intensity is increased (in this example, MA is about about). From 600a.u. to about 4500a.u.), the ECL profiles of MA and MP can be clearly distinguished. This allows MA to be detected without false positives in a single ECL measurement without the need for other analyses.

Here, the difference in ECL profile between MA and MP caused by the ECL measurement in this embodiment will be described. By synchronously capturing the ECL signal captured in the lock-in amplifier and the AC signal output from the function generator, only the ECL response generated by the application of the AC potential can be detected. Further, since other noise components are removed by the low-pass filter in the lock-in amplifier, a high-sensitivity target component can be achieved. In addition, the signal processing by the lock-in amplifier can perform the same processing and analysis by the calculation by the personal computer.

Also, the almost disappearance of the 1.2V peak in the ECL profile of MP is due to the chemical kinetics of MA and MP and Ru (bpy) 32+.
FIG. 6 shows the correlation between the ECL intensities of MA and MP due to the difference in the frequency of the alternating current to be modulated, and shows a graph in which (A) is 1.15 V and (B) is 1.45 V. As the conditions for this measurement, a detection solution containing 100 μM Ru (bpy) 32+ in a phosphate buffer solution (PBS; pH 9.0) in which 100 μM MA (a) or MP (b) is present is used. DC potential sweep rate 20 mV / s; measured at AC amplitude 50 mV.

The dependence of the ECL intensity on the superimposed frequency at 1.15V and 1.45V is such that at 1.15V, a strong and stable ECL signal is observed in MA between 5Hz and 200Hz, and then gradually decreases. On the other hand, the ELC intensity of MP decreases rapidly as the modulation frequency is increased, and is observed only slightly at a frequency of 100 Hz. This is because the dependence of the ECL intensity on the modulation frequency is related to the chemical reaction rate. As described above, the reaction rate of the reaction formula (3) is relatively fast. Therefore, in MA, the ECL reaction is performed even when the AC frequency is high. Is catching up, and it is considered that the ECL signal can be detected. On the other hand, in the case of MP, since the reaction rate of the reaction formula (6) is slow, the ECL reaction cannot keep up with the rapid change of the AC potential, and apparently, the change of the ECL signal due to the application of the AC potential cannot be observed, and 1.15V. Then, it is considered that light emission does not occur.

In contrast, the 1.45V ECL was observed only with MP. The ECL intensity is observed relatively stably at a modulation frequency of 5 Hz to 100 Hz. This is due to the high reaction rate of the reaction formula (8).

This result shows that selective detection of MA and MP can be achieved by modulated ECL measurements. It also shows that 100 Hz is appropriate as the frequency of alternating current to be modulated. Here, when the modulation frequency decreases from 100 Hz, the ECL of MP is observed at 1.15 Hz, while when it increases, the ECL intensity gradually decreases at 1.15 V and at 1.45 V. It was also shown that the ECL intensity of the was also reduced.

FIG. 7 shows the correlation of ECL intensity due to the difference in the amplitude of the modulated AC for MA, and the large graph shows the ECL intensity chart during the potential sweep due to the difference in amplitude (10 mV (71), 20 mV (72), 50 mV (73), 100 mV (74)), small graph (75) is a graph showing the correlation between amplitude and ECL intensity at 1.15 V. As the conditions for this measurement, a detection solution containing 100 μM Ru (bpy) 32+ in a phosphate buffer solution (PBS; pH 9.0) containing 100 μM MA was used, and the DC potential sweep rate was 20 mV /. s; The AC frequency was set to 100 Hz and the measurement was performed by hand.

As shown in the graph, the ECL intensity increases dramatically with an increase in AC amplitude up to 100 mV, and gradually increases above 100 mV. In addition, increasing the AC amplitude resulted in a wider peak width of the potential-ECL curve. For example, when it is set to 100 mV (74), an ECL signal is observed from 0.9 V to about 1.45 V. Therefore, if the AC amplitude is set too large, there is a concern that it will be difficult to distinguish between MA and MP. Further, if the setting is too small, the absolute intensity of ECL is low, so 50 mV was selected as an appropriate amplitude for MA detection.

FIG. 8 is a graph showing the correlation of ECL intensity with MA concentration, and the large graph is a chart of ECL intensity by modulated potential sweep due to the difference in concentration (0 nm (81), 100 nm (82), 500 nm (83), 1000 nm. (84), 2500 nm (85)), a small graph is Graph 86 showing the correlation between MA concentration and ECL intensity. The conditions for this measurement were DC potential sweep speed; 20 mV / s; AC amplitude; 50 mV, frequency: 100 Hz. In addition, a sample obtained by adding MA to the urine of a healthy person was used.

The ECL intensity at the peak of 1.15V showed a good correlation showing a linear shape proportional to the MA concentration at 0.10 μM to 2.5 μM. In this test result, r2 = 0.998. The detection limit of MA was 0.050 μM (7.5 ng / ml) for a 5 ml urine sample.

The embodiment is an example, and in addition to MA, ECL by the modulated potential sweep of the present invention enables selective detection of various substances.

11. Function generator 12. Potentiometer 13. ECL cell 14. Dark box 15. Photomultiplier tube 16. Lock-in amplifier 17. Preamplifier 18. A / D converter 19. computer

Claims (8)

A means for applying a continuous sine wave having a frequency in the range of 75 to 150 Hz as a sweep potential to an electrode in contact with the sample,
A means for simultaneously acquiring an emission signal obtained from the sample to which the sweep potential is applied and an AC cycle of the sweep potential, and
A means for extracting an AC component of the emission signal by performing arithmetic processing using the acquired emission signal and the AC cycle.
The AC component is referred to as a pattern of methamphetamine , and a means for detecting the methamphetamine ,
A drug detection system characterized by comprising. The drug detection system according to claim 1, wherein the sample is a sample selected from urine, saliva, and blood. The drug detection system according to any one of claims 1-2 , further comprising a means for removing a noise component from the emission signal. The drug detection system according to any one of claims 1-3 , wherein the amplitude of the continuous sine wave is in the range of 20 to 100 mV. A step of applying a continuous sine wave having a frequency in the range of 75 to 150 Hz as a sweep potential to the electrode in contact with the sample, and
A step of simultaneously acquiring an emission signal obtained from the sample to which the sweep potential is applied and an AC cycle of the sweep potential, and
A step of performing arithmetic processing using the acquired emission signal and the AC cycle to extract an AC component of the emission signal, and a step of extracting the AC component of the emission signal.
The step of referencing the AC component with the pattern of methamphetamine and detecting the methamphetamine , and
A drug detection method comprising. The drug detection method according to claim 5 , wherein the sample is a sample selected from urine, saliva, and blood. The drug detection method according to any one of claims 5-6 , further comprising a means for removing a noise component from the emission signal. The drug detection method according to any one of claims 5-7 , wherein the amplitude of the continuous sine wave is in the range of 20 to 100 mV.

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