TWI604187B - Surface-enhanced raman spectroscopy for rapid detection of active ingredients of pesticide products and pesticide residues in agricultural products - Google Patents

Description

Surface enhanced Raman spectroscopy method for testing pesticide products or pesticide residues in crops

The invention relates to a pesticide testing method, in particular to a Raman spectroscopy method for detecting pesticides by using Surface-Enhanced Raman Spectroscopy (SERS) combined with special material concentration technology.

The use of pesticide products in Taiwan's exquisite agriculture has hidden many problems of excessive pesticide residues. In the past few years, pesticide residues have been detected in rice, tea and various fruits and vegetables, and how to establish pesticide residues on crops. Testing and monitoring mechanisms has become a very important issue.

The current instrument for detecting the composition and concentration of pesticides is liquid chromatography tandem mass spectrometer (LC/MS-MS) or gas chromatography tandem mass spectrometer (GC/). MS-MS), which has the advantage of high detection sensitivity, but its disadvantage is that it can only be carried out in the laboratory, and it takes a long time, especially the instrument needs to use the QuEChERS method before the test (Quick, Easy, Cheap, Effective, Rugged, Safe) extracts and purifies the sample. This QuEChERS method costs approximately 2 In an hour, the required sample liquid can be taken from the sample, so it is difficult to monitor the first time and block the problem of pesticide residues.

Different from the mass spectrometer test in the laboratory, Taiwan Patent No. M506286 discloses another finished pesticide detection device, which uses the SERS substrate with Raman spectrometer to achieve the purpose of quickly detecting the active ingredients of the finished pesticide. In general, the most critical factor in the success or failure of Raman spectroscopy is the intensity of the Raman signal. In order to enhance the Raman signal, before the Raman signal is obtained, the sample to be tested will first undergo a laser pre-irradiation procedure, that is, the laser irradiation time is increased by about 2-3 seconds to enhance the pesticide molecules and the SERS substrate. The degree of adsorption of the metal structure, thereby increasing the intensity of the Raman signal.

On the other hand, patents such as CN104749159 and CN104931483, which are applied in China, also disclose similar Raman spectroscopy methods for the application of pesticide residues. In order to enhance the intensity of the Raman signal, the patents are prepared by mixing the treatment liquid of the sample to be tested with a metal nanomaterial material enhancer (for example, silver or gold nanomaterial), and then irradiating the laser light. To get enough Raman spectra.

Different from the prior art, the present invention provides another method for enhancing the Raman signal when detecting pesticides or chemicals, mainly combining traditional surface enhanced Raman spectroscopy and original concentration technology to the active ingredients of pesticides. And the rapid detection of pesticide residues in crops, by which special concentration technology can effectively enhance the intensity of Raman signals, greatly improving detection sensitivity and reliability.

Specifically, the surface enhanced Raman spectroscopy method of the present invention comprises the following steps: (a) extracting and purifying a sample to be tested to form a sample test solution; (b) extracting the sample after purification Detecting droplets attached to a SERS substrate to adsorb chemical molecules of the sample liquid on the SERS substrate; (c) continuing to deposit a highly volatile organic solvent on the SERS substrate having the chemical molecules adsorbed thereon, So that the chemical molecules are redissolved in the organic solvent to float the surface of the SERS substrate; (d) irradiating the SERS substrate with light to cause the organic solvent on the SERS substrate to be volatilized by heat, leaving chemical molecules behind Gradually concentrating to a concentrated region; (e) illuminating the concentrated region with higher energy laser light so that chemical molecules in the concentrated region are more deeply adsorbed on the SERS substrate, and the nano metal material therein Tightly combined to form a spectral measurement region of the solid phase; and (f) focusing on the spectral measurement region with laser light for Raman spectroscopy.

Preferably, the extraction solvent used for the extraction in the step (a) is selected from the group consisting of acetone, methanol, acetonitrile and an acetic acid-containing acetonitrile solution.

Preferably, the purification performed in the step (a) is such that the extracted liquid to be tested sequentially passes through a purification column and a filter membrane of one micron pore size.

Preferably, the highly volatile organic solvent used in the step (c) is diluted with at least one of acetone, methanol and ethanol.

Preferably, the highly volatile organic solvent is diluted with any of deionized water, acetonitrile and methanol.

Preferably, the light-based infrared light used in the step (d) has a wavelength between 760 nm and 2000 nm, and the output power of the light source is greater than 1 mW; and the wavelength of the infrared light used in the step (e) is between 760 nm and 1500 nm. And the output power of the light source is not more than 500mW.

Preferably, the laser light used in step (e) is a single wavelength of laser light.

Viewed from another angle, the present invention provides a chemical concentration method comprising the following steps: (a) attaching a sample to a metal substrate (such as a SERS substrate or other pure metal substrate) to The chemical molecules of the sample liquid are adsorbed on the metal substrate; (b) continuing to adhere to the metal substrate adsorbed with the chemical molecules by a highly volatile organic solvent (for example, an acetone solution diluted with deionized water) The chemical molecules are redissolved in the organic solvent to float the surface of the metal substrate; and (c) the organic solvent on the metal substrate is volatilized by heat, and the remaining chemical molecules are gradually concentrated to a concentrated region.

Preferably, step (c) is irradiated onto the metal substrate with lower energy infrared rays to rapidly evaporate the organic solvent on the metal substrate.

Preferably, the chemical concentration method further comprises after step (c), The concentrated region is irradiated with higher energy infrared laser light to more closely adsorb the chemical molecules on the metal substrate.

This chemical concentration method can be applied to Raman spectroscopy, but is not limited thereto, and can be applied to other fields.

1‧‧‧ Surface-enhanced Raman scattering substrate

11‧‧‧矽 substrate

12‧‧‧Metal nano or micro structure

2‧‧‧Laser light source

3‧‧‧Raman Spectrometer

4‧‧‧ Sample test solution

41‧‧‧Chemical molecules (pesticide molecules)

5‧‧‧Organic solvents

L1‧‧‧Infrared

L2‧‧‧Infrared

The first figure is a simplified schematic of the Raman spectroscopy detection system of the present invention.

The second figure is a schematic flow chart of the Raman spectroscopy detection method of the present invention.

The third to tenth drawings show the process of concentrating a chemical substance by the present invention using the SERS substrate 1 as a carrier.

The eleventh figure is a Raman detection spectrum of the finished pesticide Fenthion using the method of the present invention.

The twelfth figure is a Raman detection spectrum of the finished pesticide plus Carbaryl using the method of the present invention.

The thirteenth image is a conventional method for detecting the Raman detection spectrum of the finished pesticide Triazophos, in which the molecules dropped on the SERS substrate are not subjected to the reconstitution concentration process as shown in Figures 5 to 10.

The fourteenth figure is a Raman detection spectrum of the finished pesticide Triazophos by the method of the present invention, wherein the molecules dropped on the SERS substrate are subjected to a reconstitution and concentration process.

The fifteenth figure uses the method of the present invention to detect the Raman detection spectrum of the residual triazophos and the pesticide Fenthion in the wet rice.

The first and second figures show a preferred embodiment of the pesticide detection system and method of the present invention, wherein the pesticide detection method is mainly a Surface-Enhanced Raman Scattering Substrate (SERS). The substrate is used as a detection vehicle, and is combined with a laser light source 2 and a Raman spectrometer to achieve on-site pesticide detection. It can also be called surface enhanced Raman spectroscopy. The SERS substrate 1 includes a germanium substrate 11 having an area of 2.2 mm x 2.2 mm and a metal nano or micro structure 12 grown on the germanium substrate 11 by physical vapor deposition techniques, wherein the metal may be selected from gold. Materials such as silver and copper, such as silver nanopillars having a thickness of 320 nm.

Referring to the second figure, the surface enhanced Raman spectroscopy method of the present invention can be roughly divided into three stages: pretreatment, concentration and measurement. Wherein, the pre-treatment stage can be carried out by the simple extraction and purification (step 201) method described in the present invention, or by the relatively time-consuming conventional QuEChERS method. The concentration stage is mainly composed of four steps of static drying (step 202), molecular reconstitution (step 203), solvent evaporation (step 204), and adhesion bonding (step 205) included in the dotted line frame of the second figure; The measurement phase is mainly performed by measuring the Raman scattering spectrum (step 206), as described in detail below. Specifically, the surface enhanced Raman spectroscopy method of the present invention comprises the following steps: First, a sample to be tested (for example, a pesticide) The product or crop) is subjected to extraction and purification (step 201) to form a sample test solution. In this step, the sample may be pulverized or directly taken to the appropriate sample amount, and then added to the extraction solvent to be mixed by shaking or shaking. The extraction solvent may be selected from the group consisting of acetone, methanol, acetonitrile, and an organic solvent such as an acetic acid-containing acetonitrile solution. Next, 20 mL to 0.2 mL of the extract is taken and passed through a purification column and a micron-diameter filter membrane for purification to obtain a sample test liquid to complete the pretreatment stage.

Next, as shown in the third figure, 10 μL to 0.2 μL of the sample liquid 4 after extraction and purification is attached to a SERS substrate 1. After standing to dry (step 202), the chemical molecules 41 of the sample test solution 4 will be dispersed and adsorbed on the SERS substrate 1 in combination with the metal nano or microstructure 12, as shown in the fourth figure.

As shown in the fifth figure, a highly volatile organic solvent 5 is continuously applied to the SERS substrate 1 on which the chemical molecules 41 are adsorbed, so that the chemical molecules 41 are re-dissolved in the organic solvent 5 (step 203). The surface of the SERS substrate 1 is floated as shown in the sixth figure. Among them, the highly volatile organic solvent is made up of acetone, methanol and ethanol. At least one of them is diluted, and the diluent may be diluted with any one of deionized water, acetonitrile and methanol.

Then, as shown in the seventh figure, the infrared light L1 having a large light beam but low energy is first irradiated onto the SERS substrate 1 to cause the organic solvent 5 on the SERS substrate 1 to be volatilized by heat (step 204), such as the eighth. As shown in the figure, the remaining chemical molecules 41 are gradually concentrated to a concentrated area (not labeled) as shown in the ninth figure. Wherein, the wavelength of the infrared light L1 can be between 760 nm and 2000 nm, and multiple wavelengths of infrared light can be selected, and the output power of the light source is greater than 1 mW, so as to rapidly evaporate the organic solvent.

Subsequently, referring to the ninth and tenth views, the concentrated region is irradiated with the infrared light L2 having a small beam but a higher energy, so that the chemical molecules 41 in the concentrated region are adsorbed deeper on the SERS substrate 1 with the nanometer therein. The metal material is tightly bonded (step 205) to form a spectral measurement zone (not labeled) of the solid phase to complete the concentration phase. The wavelength of the infrared light L2 may be between 760 nm and 1500 nm, preferably using a single wavelength of infrared laser light, and the output power of the light source is not more than 500 mW, in order to strengthen the bonding strength between the chemical molecule and the nano metal material.

Referring to the first figure, after the concentration phase is completed, the measurement phase can be entered, that is, the laser light of the laser source 2 is focused on the spectral measurement area, and the Raman spectrum measurement is started. When the chemical molecules (pesticide molecules) concentrated and adsorbed on the SERS substrate are laser-focused to excite the Raman spectrum, the Raman spectra are different by the difference in functional structure, and the chemical molecules are judged accordingly. structure. Wherein, the wavelength of the laser light may be selected from the range of visible light or near-infrared light such as 1064 nm, 785 nm, 633 nm, 532 nm or 514 nm, and the Raman Shift measurement range of the Raman spectrum is between 200 cm-1 and 4000cm-1.

In summary, when the sample liquid 4 is attached to the SERS substrate 1, the molecules of the test object (that is, the pesticide molecules) in the test liquid will compete with other non-substance molecules in the test liquid for adsorption to the SERS. After the surface of the substrate 1 is left to dry (step 202), the molecules are widely distributed and adsorbed on the SERS substrate 1. Then, the adsorbed analyte molecules are reconstituted by a solution mixed with a specific solvent ratio (step 203), and concentrated by light of appropriate power (steps 204-205), whereby a chemical concentration method may have Concentrate on pesticide molecules and enhance Raman spectroscopy signals. It is worth noting that the solution used in reconstitution (that is, the highly volatile organic solvent) must have the characteristics of being volatile and re-dissolving the molecules of the analyte, and protecting the molecules of the analyte from being absorbed in the light concentration process. Excessive laser light destroys the molecular structure, causing the signal strength to decrease or misjudge.

By the above method, the crop sample is attached to the SERS substrate for Raman spectroscopy after simple extraction and rapid purification and concentration steps, and can be completed within ten minutes on the spot, and the type and content of residual chemical substances are read by the computer. It can greatly improve the efficiency of pesticide testing and block unqualified pesticides in the first time.

A few examples will be detailed below:

Experiment 1: Detection of the finished pesticide Fenthion

Please refer to the eleventh figure of the finished pesticide Fenicide pine Raman detection spectrum, in which the test sample is 50% concentration, the brand Libashan Fenicide Loose Emulsion (organophosphorus). First, the test sample is diluted to 100 ppm by acetone, and after swirling for 30 seconds, 0.5 ml of the mixed solution is passed through a purification column containing an appropriate amount of C18 and PSA powder, and the purification column is connected in series. A 0.2 μm pore size Nylon filter was used for filtration. Subsequently, 2 μl of the purified sample was spotted on a SERS substrate using a micropipette and waited for drying. Wherein, the SERS substrate has a germanium substrate having an area of 2.2 mm x 2.2 mm and a silver nano column structure having a thickness of 320 nm deposited on the germanium substrate. The dried pesticide molecules will be adsorbed on the silver nano column structure, and then 2μl of acetone: deionized water (1:1, v/v) mixture (that is, high volatile organic solvent) is added to the SERS substrate to make the pesticide. Molecular reconstitution. Subsequently, the SERS substrate is irradiated with infrared laser light having a wavelength of 808 nm and a power of 200 mW to 300 mW to rapidly evaporate the organic solvent, leaving a concentrated pesticide molecule. Next, the SERS substrate is irradiated with infrared laser light having a wavelength of 785 nm and a power of 100 mW, and the concentrated pesticide molecules are further adsorbed on the silver nano column structure to complete the preparation of the sample. Finally, in the Raman spectroscopy measurement, the laser source uses a wavelength of 785 nm, a power of 80 mW, a lens magnification of 4X, an integration time of 500 ms, and an average number of times of 32 times. According to the measurement results, the main characteristic peaks of the Raman detection spectrum of the pesticides of Fenicides were located at 1044 cm-1, 1224 cm-1, 1569 cm-1, etc., which were consistent with the spectra of the standard samples. It is worth noting that 520 in the Raman detection spectrum The cm-1 is the Raman signal of the substrate itself under the SERS substrate, which is not caused by pesticide molecules. The overall detection time of this test method is less than 10 minutes, and the effective component of the finished pesticide can be determined qualitatively immediately.

Experiment 2: Detection of finished pesticides plus Poly

Please refer to Figure 12 for the Raman detection spectrum of the finished pesticide plus Carbaryl. The test sample is an 85% strength, wettable powder type of Poly-Pesticide (Aminoformate). First, the test sample is diluted to 100 ppm by acetone, and after swirling for 30 seconds, 0.5 ml of the mixed solution is passed through a purification column containing an appropriate amount of C18 and PSA powder, and the purification column is connected in series. A 0.2 μm pore size Nylon filter was used for filtration. Subsequently, 2 μl of the purified sample was spotted on a SERS substrate using a micropipette and waited for drying. Wherein, the SERS substrate has a germanium substrate having an area of 2.2 mm x 2.2 mm and a silver nano column structure having a thickness of 320 nm deposited on the germanium substrate. After drying, the pesticide molecules are adsorbed on the silver nano column structure, and the mixture of 2 μl of methanol: deionized water (1:1, v/v) (that is, a highly volatile organic solvent) is attached to the SERS substrate to make the pesticide molecule. Reconstituted. Subsequently, the SERS substrate is irradiated with infrared laser light having a wavelength of 808 nm and a power of 200 mW to 300 mW to rapidly evaporate the organic solvent, leaving a concentrated pesticide molecule. Next, the SERS substrate is irradiated with infrared laser light having a wavelength of 785 nm and a power of 100 mW, and the concentrated pesticide molecules are further adsorbed on the silver nano column structure to complete the preparation of the sample. In the Raman spectroscopy measurement, the laser source uses a laser wavelength of 785 nm, a power of 100 mW, a lens magnification of 4X, an integration time of 500 ms, and an average number of times of 32 times. According to the measurement results, the main characteristic peaks of the Raman detection spectrum of the poly-protective pesticide molecules are located at 1385 cm-1 and 1420 cm-1, which are consistent with the standard sample spectrum. Similarly, the Raman signal at 520 cm-1 in the Raman detection spectrum is the substrate itself in the lower layer of the SERS substrate, not the pesticide molecule.

Experiment 3: Comparison of the detection of the finished pesticides

Please refer to the Raman detection spectrum of the finished pesticide Triazophos in the thirteenth figure, in which the pesticide product is diluted to 10 ppm by acetone, and after swirling for 30 seconds, 0.5 ml of the mixture is passed through a purification column. The purification column contains an appropriate amount of C18 and PSA powder, and the purification column is connected in series with a 0.2 μm aperture Nylon Filter the filter for filtration. Subsequently, 2 μl of the purified sample was spotted on a SERS substrate by using a micropipette having a ruthenium substrate having an area of 2.2 mm x 2.2 mm and a silver nanocolumn structure having a thickness of 320 nm deposited on the ruthenium substrate. . After drying, the pesticide molecules will adsorb on the silver nanocolumn structure. At this time, if it is directly measured by a Raman spectrometer after drying, a Raman spectrum as shown in Fig. 13 can be obtained. On the other hand, if the pesticide molecules continue to complete the reconstitution concentration procedure after drying and then perform Raman spectroscopy, a Raman spectrum as shown in Fig. 14 can be obtained. In other words, in the former experiment, the sample test solution was not subjected to a reconstitution and concentration process, and it can be used as a reference for those who have undergone reconstitution and concentration procedures.

In detail, if it is dried, 2μl of methanol: deionized water (1:1, v/v) mixture (that is, high volatile organic solvent) is added to the SERS substrate to reconstitute the pesticide molecules, and then illuminate. concentrate. Among them, in the concentration process, the SERS substrate is irradiated with infrared laser light having a wavelength of 808 nm and a power of 200 mW to 300 mW, so that the organic solvent is quickly volatilized, leaving concentrated pesticide molecules. Next, the SERS substrate is irradiated with infrared laser light having a wavelength of 785 nm and a power of 100 mW, and the concentrated pesticide molecules are further adsorbed on the silver nano column structure to complete the preparation of the sample. Finally, a Raman spectrometer was placed for SERS spectroscopy to obtain a Raman spectrum as shown in Fig. 14. The laser source of Raman spectroscopy was 785 nm, the power was 80 mW, and the lens magnification was 4X. The integration time is 500ms and the average number of times is 32. According to the measurement results, the main characteristic peaks of the Raman spectra of the three falling pine pesticide molecules are located at 983 cm-1, 1004 cm-1, 1410 cm-1, 1548 cm-1, and 1599 cm-1, which are consistent with the standard sample spectrum. More importantly, the signal intensity of the Raman spectrum of the fourteenth figure is nearly five times stronger than that of the thirteenth figure. It can be seen that the reconstitution and concentration method of the present invention can enhance the Raman signal and greatly improve the detection sensitivity.

Experiment 4: Detection of multiple pesticide residues in crops

The fifteenth figure shows the Raman detection spectrum of pesticides such as Triazophos and Fenthion remaining in wet rice. In general, after drying and dehulling, the concentration of pesticide residues in brown rice and white rice will be much less than 5 ppm. Therefore, in this experiment, the test sample is added with 5 ppm of trilopine in wet rice. (Triazophos) and 5 ppm of Fenthion. First, after mixing the above 10 g test sample with 10 ml of acetone, hand-shake for 30 seconds and take out 1 ml of the extraction solution through a purification column, and the PSA, C18, MgSO4, and GCB powders are simultaneously added to the purification column to obtain a purification solution. After that, 2 μl of the solution (that is, the sample test solution) was dropped on the SERS substrate (320 nm; silver nano column structure) as described above, and waited for drying until the pesticide molecules were adsorbed on the silver nano column structure, and then 2μl of methanol: deionized water (1:1, v/v) mixture (that is, high volatile organic solvent) is attached to the SERS substrate to re-dissolve the pesticide molecules, and firstly use infrared ray with a wavelength of 808nm and a power of 200mW~300mW. The SERS substrate is irradiated with light to rapidly evaporate the organic solvent, leaving concentrated pesticide molecules, and then irradiating the SERS substrate with infrared laser light having a wavelength of 785 nm and a power of 100 mW, so that the concentrated pesticide molecules are further deeply adsorbed on the silver nano column structure. , the preparation of the finished product. Finally, Raman spectroscopy is performed, in which the laser light system adopts a wavelength of 785 nm, a power of 100 mW, a lens magnification of 4×, an integration time of 500 ms, and an average number of times of 32 times. It can be seen from the spectrogram that the main characteristic peak is located at the main position of the standard molecule of 983cm-1, 1004cm-1, and 1044cm-1, 1224cm-1, and 1569cm-1 are the main positions of the standard molecules of Fenicide. The intensity of the Raman peak can be used as the basis for the semi-quantitative determination of pesticide residues. Therefore, the present invention can also be used to detect a plurality of pesticide residues on crops.

In any event, anyone can obtain sufficient teaching from the description of the above examples, and it is understood that the present invention is indeed different from the prior art, and is industrially usable and progressive. It is the invention that has indeed met the patent requirements and has filed an application in accordance with the law.

1‧‧‧ Surface-enhanced Raman scattering substrate

11‧‧‧矽 substrate

12‧‧‧Metal nano or micro structure

2‧‧‧Laser light source

3‧‧‧Raman Spectrometer

Claims (10)

A surface enhanced Raman spectroscopy method comprising the following steps: (a) extracting and purifying a sample to be tested to form a sample test solution; (b) attaching the sample to the sample after extraction and purification a SERS substrate for adsorbing chemical molecules of the sample liquid on the SERS substrate; (c) continuing to immerse the highly volatile organic solvent on the SERS substrate on which the chemical molecules are adsorbed to reconstitute the chemical molecules In the organic solvent, the surface of the SERS substrate is floated; (d) the SERS substrate is irradiated with light to cause the organic solvent on the SERS substrate to be volatilized by heat, and the remaining chemical molecules are gradually concentrated to a concentrated region. (e) further irradiating the concentrated region with laser light such that chemical molecules in the concentrated region are more deeply adsorbed on the SERS substrate, thereby forming a spectral measurement region of the solid phase; and (f). The illuminating light is focused on the spectral measurement area for Raman spectroscopy. As described in claim 1, the extraction solvent used in the step (a) is selected from the group consisting of acetone, methanol, acetonitrile and an acetic acid-containing acetonitrile solution. As described in the first aspect of the patent application, the purification performed in the step (a) is such that the liquid to be tested after the extraction passes through a purification column and a filter membrane of a micrometer aperture. The method of surface-enhanced Raman spectroscopy according to claim 1, wherein the highly volatile organic solvent used in the step (c) is diluted with at least one of acetone, methanol and ethanol. . The surface enhanced Raman spectroscopy method according to claim 4, wherein the diluent for diluting to the highly volatile organic solvent comprises any one of deionized water, acetonitrile and methanol. The surface enhanced Raman spectroscopy method according to claim 5, wherein the light-based infrared light used in the step (d) has a wavelength of 760 nm to 2000 nm, and an output power of the light source is greater than 1 mW; The wavelength of the laser light used in the step (e) is between 760 nm and 1500 nm, and the output power of the light source is not more than 500 mW. The surface enhanced Raman spectroscopy method according to claim 6, wherein the infrared ray used in the step (e) is a single wavelength laser light. A method for concentrating a chemical substance, comprising the following steps: (a) attaching a sample to a metal substrate to adsorb chemical molecules of the sample liquid on the metal substrate; (b) continuing to be highly volatile The organic solvent is dropped on the metal substrate on which the chemical molecule is adsorbed, so that the chemical molecule is redissolved in the organic solvent to float the surface of the metal substrate; and (c) the organic solvent on the metal substrate It is volatilized by heat, and the remaining chemical molecules are gradually concentrated to a concentrated area. The method of concentrating a chemical substance according to claim 8, wherein the step (c) is irradiated onto the metal substrate with a lower energy infrared ray to rapidly evaporate the organic solvent on the metal substrate. The method for concentrating a chemical substance according to claim 9, further comprising, after the step (c), irradiating the concentrated region with a higher energy infrared laser light to more closely adsorb the chemical molecules on the metal substrate. .