Progress 04/01/18 to 03/31/22
Outputs
Target Audience:PD, Dr. Dan Du, is supervising the PhD student, Xiaofan Ruan and co-supervising PhD student Eunice Kwon. She was invited to give several lectures on "Nanobiosensors for food safety and environmental application" for graduates in Food Science, Chemical Engineering and Materials Engineering from 2019 to 2021. She covers nanoscale-based sensing mechanisms, signal amplification schemes, and smart sensors for the reliable and cost-effective detection of pathogens and pesticides in food. Dr. Du gives her suggestions to both students on the development of multiplexed biosensors, evaluation of the selectivity, sensitivity, and repeatability of the biosensors for simultaneous detection of multiple pesticides in juices, fruits, vegetables, and groundwater. Co-PD, Prof. Bernie Van Wie, is supervising the efforts of PhD student Eunice Kwon and co-supervising the efforts of PhD student, Xiaofan Ruan whose major advisor is Dr. Du and Dr. Lin. Typically, Van Wie gives feedback at bi-weekly team meetings to Kwon and Ruan and has been interacting with Kwon on her first publication, NASA grant application and poster presentation. Co-PD, Prof. Yuehe Lin, is teaching a graduate class on Nanoscience and Nanotechnology in the spring semester in 2019 and 2021. Lin includes nanotechnology for agricultural and food systems in his class. He covers nanomaterials synthesis, characterization, nanosensor mechanisms and applications in detection of pathogens, insects, diseases, chemicals, and contaminants in food, water and the agricultural production environment. Dr. Lin is supervising PhD student, Xiaofan Ruan, especially on the 3D printing technology and the design of electrochemical biosensors. Co-PD, Prof. Meijun Zhu covers on validating the biosensing approach with HPLC analysis in real samples. She provides feedback on sample preparation and comparison with HPLC results in standard solution, spikes samples, and raw samples. Eunice Kwon, PhD student, received a NASA Space Grant Fellowship in Science and Engineering. Based on what she has learned from this project, she proposed research to develop multiplex test strips to detect several kinds of pesticides simultaneously with advantages of this method, such as rapid detection in agricultural fields and cost-effectiveness of peroxidase-like nanoparticles. With this fellowship, she attended the AIChE 2019 annual meeting in Orlando, FL. In 2020, she presented her results at the virtual AIChE 2020 annual meeting. She received the women in chemical engineering (WIC) travel award and an honorable mentioned student award at the student competition in the environmental sensors session. Xiaofan Ruan is a Ph.D. student in the School of Mechanical and Materials Engineering. He is studying nanomaterials and devices for biosensing with interdisciplinary application of 3D-printing for food safety and environmental applications. He presented his results at the Graduate and Professional Students Association (GPSA) Academic Showcase Research Exposition at WSU each year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two PhD students (Eunice Kwon and Xiaofan Ruan) have been trained on this project. Both of them learned about nanomaterials synthesis, characterization, and signal amplification mechanism. They understood immunoassay principles, mechanisms behind, and techniques for developing lateral flow test strips, along with optimization procedures, determination of the analytical linear range, the limit of detection, and recovery. They came to learn new 3D printing technology and designed multiplexed electrochemical biosensing devices and applied them for simultaneous detection of multiple pesticides. To validate our device using real samples, the students learned food sample processing called the QuEChERS technique (Quick, Easy, Cheap, Effective, Rugged, and Safe). This technique is widely used for pesticide residues analysis in food matrices, including fruits and vegetables. Bi-weekly project meetings served to train them on how to organize their results and improve presentation skills. How have the results been disseminated to communities of interest?Students presented their results at the Graduate and Professional Students Association (GPSA) Academic Showcase Research Exposition at WSU each year. A PhD student, Eunice Kwon, presented her results at the AIChE 2019 Annual Meeting in Orlando, FL, and at the virtual AIChE 2020 Annual Meeting. Four paper has been published on Small 2019, Biosensors and Bioelectronics 2019, Analytica Chimica Acta 2020 and Biosensors and Bioelectronics 2021. One paper has been accepted for publication in Food Chemistry under the title "Simultaneous Detection of Two Herbicides in Fruits and Vegetables with Nanoparticle-linked Immunosorbent and Lateral Flow Immunoassays". One review article under the title "Immunoassay technologies for the simultaneous analysis of multiple pesticide residues in food and water" has been submitted to Food Chemistry. Both students Eunice Kwon and Xiaofan Ruan have passed their final PhD defense on February 1, 2022 and April 1, 2022 due to the support by this project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the past four years, we successfully completed all the proposed aims and tasks of this project. We synthesized and characterized several Pt-based bimetal nanoparticles under Aim 1; developed multiplexed immunosensor devices for simultaneous detection of pesticides under Aim 2; and validated our devices for detection of pesticides in real samples (fruits, vegetables and groundwater) under Aim 3. Major activities completed / experiments conducted Aim 1: To synthesize and characterize nanoparticle-tagged antibodies. (1) Three kinds of Pt-based nanomaterials, mesoporous Pd@Pt nanoparticles, Pd@Au nanoparticles and 2D Pt-Ni(OH)2 nanosheets were synthesized and characterized. Synthesis of Pd@Pt and Pd@Au nanoparticles begins with a Pluronic® F127 surfactant to form self-assembled micelles with the hydrophobic polypropylene oxide (PPO) facing inward and hydrophilic polyethylene oxide (PEO) facing outward. Then ionization of selected Pd, Au and Pt salts in the form of HAuCl4, Na2PdCl4 and K2PtCl4 species leads to formation of PdCl42-, AuCl4-and PtCl42- ions. In the presence of ascorbic acid (AA) as a reducing agent, PdCl42 or AuCl4- reduction occurs at a higher rate than that of PdCl42 and therefore a NP forms where Pd is the dominate species in the core.Transmission electron microscopy (TEM), X-ray diffraction (XRD) and energy dispersive spectroscopy (EDS) have been used to characterize the morphologies of the nanomaterials. We observed that Pt@Pd and Pt@Au nanoparticles showed a nearly spherical uniformwith an average particle size of ~50 nm. They consist of a branched structure, but are nearly spherical,with an approximate 9:1 ratio of atomic Pt:Pd. Pt-Ni(OH)2 nanosheets were obtained via a two-step microwave method. Ni(OH)2 was synthesized via dissolving nickel nitrate and urea in H2O and ethylene glycol solution. H2PtCl6 and Ni(OH)2 were dissolved in a vial containing 5 mL EG and 5 mL H2O. The TEM images of Pt-Ni(OH)2 exhibited uniform distribution of Pt nanoclusters on Ni nanosheets. The size distribution of Pt nanoclustersis 2 nm. (2) Pt-based nanoparticles showed peroxidase-like catalytic activity which is superior to that of monometallic Pt nanoparticles and enzyme HRP. We observed a low Km with an H2O2 substrate for mesoporous Pt@Pd nanoparticles of 0.053mM which is two to three orders of magnitude less than the 11.1 mM value for Pt nanopowders and 3.70 mM for HRP. Both nanomaterials displayed high peroxidase-like activity to catalyze the H2O2-,3',5,5'-tetramethylbenzidine (TMB) redox reaction while maintaining stability over a 1-11 pH range and 4-90 °C temperature range. The mesoporous Pd@Pt NPs also have higher thermo- and pH-stability than HRP. Aim 2: To develop a multiplex immunosensing system and establish a nanoparticle electrochemical detection approach. (1) Development of a two-channel multiplex immunosensor device. The strips were constructed to have a sample pad, conjugation pad, nitrocellulose membrane, and absorbent pad on a supporting membrane card. Each channelcontained a 12 mm x 4 mm absorbent pad and a triangular sample pad. The control linewas created by dispensing goat-anti-mouse IgG antibodies. The test lines were prepared by dispensing BSA-atrazine and BSA-acetochlor respectively. Assay parametersincluding pretreatment of conjugate pads and concentration of coated antibody-nanoparticle complex. The corresponding redox reaction is monitored by differential pulse voltammetry (DPV) scanning from -0.8 to -0.1 V. The simultaneous detection of atrazine and acetochlor was 0.3 ng/mL and 3.3 ng/mL, respectively. (2) Development of two-way lateral flow immunoassays with smartphone readout for quantification of pesticides. The test lines (1 μL/cm) were prepared by dispensing 30 μL of semi-antigens of acetochlor or fenpropathrin on the NC membrane. Control lines (1 μL/cm) were prepared by dispensing 30 μL of goat anti-mouse IgG on the same NC membranes to maintain a 1 cm gap with test lines. We developed a universal detection app, Strip Scan for quantification of herbicidescontinuously.Using the integrated platform, detection of acetochlor and fenpropathrin was of 0.63 ng/mL and 0.24 ng/mL, respectively. (3) Development of windmill-like multiplexed immunosensor. A windmill-like three-channel strip was developed for simultaneous detection of three pesticides, atrazine, acetochlor, and chlorpyrifos. In the design, the three test strips in the channels shared one circular sample pad (Ø = 10 mm), and each test strip was constructed by one 15 mm × 4 mm absorbent pad, one 25 mm × 4 mm nitrocellulose membrane, and one 10 mm × 4 mm conjugate pad. Other sensor platform parts were 3D printed accessories, including a strip cutter, a strip holder, and an electrode holder. The measurement was based on competitive immunoassays. The targets of atrazine, acetochlor, and chlorpyrifos competed with BSA-haptens on the conjugation zones respectively to bind to the limited sites of corresponding antibody-Pd@Pt NPs conjugates coated on the conjugate pads. A time frame of 10 min was optimized. The detection limit of 0.22 ng/mL for atrazine, 3.1 ng/mL for acetochlor, and 0.017 ng/mL for chlorpyrifos was achieved. Aim 3: To validate the IEB device using real samples. (1) Simultaneous detection of two herbicides in fruits and vegetables. We utilized two Pd@Pt nanoparticle-amplified immunoassays for real sample assays. We achievedrecoveries of 88.5 - 114% in juice, fruit, and vegetable samples. Thelimit of detection is0.59 and 0.31 ng/mL for atrazine (ATZ) and acetochlor (ACT) respectively, and the dual-LFIA-DPV obtained the LOD of 0.27 and 0.51 ng/mL respectively for ATZ and ACT. Results were validated and compared with HPLC. (2) Fruit and vegetable sample preparation. Each fruit or vegetable sample was blended with a blender. The extracted samples were centrifuged at 12,000 rpm for 10 min. After adding the pesticides, the samples were extracted with 70% methanol for 10 min at RT with shaking. The extracted samples were centrifuged at 12,000 rpm for 10 min. (3) Detection of atrazine and acetochlor in real samples with HPLC. HPLC sample analysis was carried out using a Shimadzu LC20 with a UV detector at 215 nm. The analytical column was an Agilent ZOPBAX Eclipse XDB-C18 (4.6 mm x 250 mm, 5 μm) with 35°C of column temperature. The mobile phase was created with a methanol-water solution (75-25, v/v) adjusted to pH 3 with 85% phosphoric acid. A 1.0 mL/min flow rate was used. a) Sample preparation (QuEChERS methods). Sample preparation used QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method with modifications to adjust to our lab conditions. First, 30 ml of a juice sample was centrifuged at 8000 rpm for 15 min and filtrated with a syringe filter. NaCl (6 g) was added to 20 ml of the centrifuged and filtrated sample and mixed for 1 min. Then, 20 ml of acetonitrile containing 1% acetic acid (v/v) was added to the sample and mixed for 1 min. Next, the sample was held at room temperature for 1 h, followed by centrifugation at 8000 rpm for 5 min. The upper phase (acetonitrile phase) was transferred to a glass vial and evaporated with a nitrogen evaporator. The evaporated samples were then dissolved into 2 ml of methanol to remove the salt. b) Detection of atrazine and acetochlor spiked in juice samples. Apple and watermelon juice samples were spiked with these two herbicides at four different concentrations 30, 50, 80, and 100 ng/mL. The presence of the herbicides in the samples was detected by HPLC with the recoveries of 95.4 - 102% (in triplicate). c) Detection of atrazine and acetochlor spiked in real samples. Fruit and vegetable samples were spiked with these two herbicides at four different concentrations. HPLC analysis resulted in recoveries between 87.7 and 120%.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
PtNi(OH)2 nanosheets amplified two-way lateral flow immunoassays with
smartphone readout for quantification of pesticides. Biosensors and Bioelectronics 142 (2019) 111498
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Single-Atom Nanozyme Based on Nanoengineered
FeNC Catalyst with Superior Peroxidase-Like Activity
for Ultrasensitive Bioassays. Small 2019, 1901485
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Mesoporous Pd@Pt nanoparticle-linked immunosorbent assay for
detection of atrazine. Analytica Chimica Acta 1116 (2020) 36e44
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Nanomaterial-enhanced 3D-printed sensor platform for simultaneous
detection of atrazine and acetochlor. Biosensors and Bioelectronics 184 (2021) 113238
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Simultaneous Detection of Two Herbicides in Fruits and Vegetables with Nanoparticle-linked Immunosorbent and Lateral Flow Immunoassays. Food Chemsitry
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Immunoassay technologies for the simultaneous analysis of multiple pesticide residues in food and water. Food Chemistry
|
Progress 04/01/21 to 03/31/22
Outputs
Target Audience:PD, Dr. Dan Du, supervises the PhD student, Xiaofan Ruan and co-supervising PhD student Eunice Kwon. She was invited to lecture on "Nanobiosensors for food safety and environmental application" for graduates in Food Science in 2021. She covers nanoscale-based sensing mechanisms, signal amplification schemes, and smart sensors for reliable and cost-effective detection of pathogens and pesticides in food. Co-PD, Prof. Bernie Van Wie, supervises the efforts of PhD student Eunice Kwon and co-supervises the efforts of PhD student, Xiaofan Ruan whose major advisor is Dr. Du. Typically, Van Wie gives feedback at twice-monthly team meetings to Kwon and Ruan and has been interacting with Kwon on her first publication, NASA grant application, and presentation. Co-PD, Prof. Yuehe Lin, teaches a graduate class on Nanoscience and Nanotechnology in the spring semester and includes nanotechnology for agricultural and food systems. He covers nanomaterials synthesis, characterization, nanosensor mechanisms, and applications to detect pathogens, insects, diseases, chemicals, and contaminants in food, water, and the agricultural production environment. Eunice Kwon, a PhD student, received a NASA Space Grant Fellowship in Science and Engineering. Based on what she has learned from this project, she proposed research to develop multiplex test strips to detect several kinds of pesticides simultaneously with advantages of this method, such as rapid detection in agricultural fields and cost-effectiveness of peroxidase-like nanoparticles. Xiaofan Ruan is a Ph.D. student in the School of Mechanical and Materials Engineering. He studies nanomaterials and devices for biosensing with the interdisciplinary application of 3D-printing for food safety and environmental applications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two PhD students (Eunice Kwon and Xiaofan Ruan) have been trained on this project. Both learned about nanomaterials synthesis, characterization, and signal amplification mechanism. This year both of them focused on real sample analysis and trained on HPLC analysis. They came to learn new 3D printing technology and designed multiplexed electrochemical biosensing devices and applied them for simultaneous detection of multiple pesticides. To validate our device using real samples, the students learned food sample processing called the QuEChERS technique (Quick, Easy, Cheap, Effective, Rugged, and Safe). This technique is widely used for pesticide residues analysis in food matrices, including fruits and vegetables. Bi-weekly project meetings served to train them on how to organize their results and improve presentation skills. How have the results been disseminated to communities of interest?Students presented their results at the Graduate and Professional Students Association (GPSA) Academic Showcase Research Exposition at WSU. One paper was published on Biosensors and Bioelectronics under the title "Nanomaterial-enhanced electrochemical immunosensor for simultaneous detection of atrazine and acetochlor on a 3D-printed platform." One review article under the title "Immunoassay technologies for the simultaneous analysis of multiple pesticide residues in food and water" is in preparation and was submitted to Trend in Analytical Chemistry. Data of HPLC results are collected and has been submitted to Food Chemistry and it is under review. Both students Eunice Kwon and Xiaofan Ruan have passed their final PhD defense on Feburary 1, 2022 and April 1, 2022 based on the support by this project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the last year of this project, we explored the feasibility of our technology to detect pesticides in fruits, vegetables, and groundwater under Aim 3. (1) Simultaneous detection of two herbicides in fruits and vegetables. We utilized two Pd@Pt nanoparticle-amplified immunoassays including a colorimetric Pd@Pt nanoparticle-linked immunosorbent assay (NLISA) and a differential pulse voltammetry (DPV) dependent on catalytic activity of Pd@Pt in a dual-lateral flow immunoassay (dual-LFIA-DPV). We achieved overall recoveries of 88.5 - 114% in juice, fruit, and vegetable samples for both immunoassays. The NLISA yielded limit of detection (LOD) of 0.59 and 0.31 ng/mL for atrazine (ATZ) and acetochlor (ACT) respectively; and the dual-LFIA-DPV obtained the LOD of 0.27 and 0.51 ng/mL respectively for ATZ and ACT. Results for both immunoassays were validated and compared with HPLC. a) Fruit and vegetable samples preparation The fruits and vegetables--apples, strawberries, corn, and cabbage--were purchased at a local market near Washington State University (Pullman, WA). Sample preparation for immunoassays was modified from a previous paper (Talanta 2012, 101, 85-90). First, each fruit or vegetable sample was blended with a blender. After adding the pesticides into the samples, the samples were extracted with 70% methanol for 10 min at RT with shaking. The extracted samples were centrifuged at 12,000 rpm for 10 min. The samples were diluted with PBS to arrive at desired concentrations. First, each fruit or vegetable sample was blended with a blender. After adding the pesticides into the samples, the samples were extracted with 70% methanol for 10 min at RT with shaking. The extracted samples were centrifuged at 12,000 rpm for 10 min. The samples were diluted with PBS for to arrive at desired concentrations. b) Pd@Pt nanoparticle-linked immunosorbent assay (NLISA) The indirect competitive ELISA-like format NLISA method follows that outlined in a previous publication (Analytica Chimica Acta 2020, 1116, 36-44). First, BSA conjugates were diluted at a 1:1000 ratio for ATZ-BSA or a 1:2000 for ACT-BSA in carbonate bicarbonate coating buffer (pH 9.6 and 0.05 M). The 50 μL of each diluted BSA conjugate solution was covered on the 96 well plate; the plate was incubated for 2h at 37 °C. After the plate was washed 3 times with PBST buffer, the coated plate was blocked by adding a 200 μL of blocking PBS buffer containing 3% BSA for ATZ and 1% BSA for ACT. Then, the plate was incubated for another 1.5 h at 37 °C. After the plate was washed 3 times with PBST, 50 μL of mixtures that contained ATZ and/or ACT as well as Ab-Pd@Pt conjugates in a 1:1 ratio was added to the 96 well plate for binding of any excess Ab-Pd@Pt. The plate was incubated for 1 h at 37 °C and washed 5 times with PBST. Afterward, 50 μL of TMB substrate was added to the plate followed by incubation for 10 min at 37 °C for an enzyme-like reaction; therefore, the color was changed to blue. Then, 50 μL of 2 M H2SO4 was added to the plate to stop the reaction and change to yellow. Lastly, absorbances for the reaction mixture were measured at 450 nm using a Cytation 5 plate reader from BioTek®. The results for NLISA yielded an LOD for ATZ of 0.59 ng/mL (10% inhibition, i.e., 90% of B/B0) with a linear range of 0.25 - 1000, an RSD of 3.30%, slope of -24.5 and R2 = 0.996 and an LOD for ACT of 0.31 ng/mL (10% inhibition) with a linear range of 0.1 - 500 ng/mL, an RSD 2.58%, slope of= -14.7 and R2 of 0.995. c) Pd@Pt nanoparticle based dual-lateral flow immunoassay (dual-LFIA-DPV) The dual-LFIA consisted of one 9 mm × 20 mm triangular sample pad, two 4 mm × 5 mm conjugate pads, two 4 mm × 30 mm nitrocellulose (NC) membranes, and two 4 mm × 15 mm absorbent pads. After the test strips are constructed, 100 μL of a sample solution is placed onto the sample pad for 10 - 15 min, allowing the sample solution to flow to the absorbent pad by capillary action. As the sample flows from the conjugate pad through the dual NC membranes, the intensity of the test line depends on the inverse proportion of the concentration of analytes in the sample.The DPV method was used as follows. First, test lines from the test strips are cut by a punchcutter connected to the device, causing the strips to fall into a chamber well containing screen-printed electrodes. After adding 80 μL of substrate solution, the chamber is covered and incubated for 2 min. The results are monitored using a CHI1030A multi-potentiostat (CH Instruments, Austin, TX) to measure DPV signals while scanning from -0.1 to -0.7 V, with an amplitude of 0.02 V and pulse width of 0.05 s. The results for the dual-LFIA-DPV show linearity with LODs of 0.27 ng/mL for ATZ and 0.51 ng/mL for ACT with respective linear ranges between 1 and 200 ng/mL and an average RSD of 4.67% and between 1 and 500 ng/mL and an average RSD over 3.75%. (2) Detection of atrazine and acetochlor in real samples with HPLC. HPLC sample analysis was carried out using a Shimadzu LC20 with a UV detector at 215 nm. The analytical column was an Agilent ZOPBAX Eclipse XDB-C18 (4.6 mm x 250 mm, 5 μm) with 35°C of column temperature. The mobile phase was created with a methanol-water solution (75-25, v/v) adjusted to pH 3 with 85% phosphoric acid. A 1 mL/min flow rate was used. a) Sample preparation (QuEChERS methods) We utilized the QuEChERS method for extraction with modifications as described here to prepare spiked juice samples. First, 30 mL of a juice sample was centrifuged at 8000 rpm for 15 min and filtrated with a syringe filter (0.45 μm). Then, 6 g of NaCl was added to 20 mL of the sample and mixed for 1 min. Next, 20 mL of acetonitrile containing 1% acetic acid (v/v) was added to the sample and mixed for 1 min. Then, the sample was held at RT for 1 h, after which it was centrifuged at 8000 rpm for 5 min. The upper acetonitrile phase was transferred to a glass vial and evaporated with a nitrogen evaporator (N-Evap 104 from Organomation Associates, Inc.). The evaporated samples were then dissolved into 2 mL of methanol to remove the salt. b) Detection of atrazine and acetochlor spiked in juice samples Apple and watermelon juice samples were spiked with these two herbicides at four different concentrations: atrazine at 30, 50, 80, and 100 ng/mL and acetochlor at 80, 100, 150, 200 ng/mL, respectively. The presence of the herbicides in the samples was detected by HPLC with the recoveries of 95.4 - 102% (in triplicate). c) Detection of atrazine and acetochlor spiked in real samples Fruit and vegetable samples were spiked with these two herbicides at four different concentrations, same as juice samples: atrazine at 30, 50, 80, and 100 ng/mL and acetochlor at 80, 100, 150, 200 ng/mL, respectively. HPLC analysis resulted in recoveries between 87.7 and 120%.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Nanomaterial-enhanced 3D-printed sensor platform for simultaneous
detection of atrazine and acetochlor. Biosensors and Bioelectronics 184 (2021) 113238
|
Progress 04/01/20 to 03/31/21
Outputs
Target Audience:PD, Dr. Dan Du, supervises the PhD student, Xiaofan Ruan and co-supervising PhD student Eunice Kwon. She was invited to lecture on "Nanobiosensors for food safety and environmental application" for graduates in Food Science in 2020. She covers nanoscale-based sensing mechanisms, signal amplification schemes, and smart sensors for reliable and cost-effective detection of pathogens and pesticides in food. Co-PD, Prof. Bernie Van Wie, supervises the efforts of PhD student Eunice Kwon and co-supervises the efforts of PhD student, Xiaofan Ruan whose major advisor is Dr. Du. Typically, Van Wie gives feedback at twice-monthly team meetings to Kwon and Ruan and has been interacting with Kwon on her first publication, NASA grant application, and presentation. Co-PD, Prof. Yuehe Lin, teaches a graduate class on Nanoscience and Nanotechnology in the spring semester and includes nanotechnology for agricultural and food systems. He covers nanomaterials synthesis, characterization, nanosensor mechanisms, and applications to detect pathogens, insects, diseases, chemicals, and contaminants in food, water, and the agricultural production environment. Eunice Kwon, a PhD student, received a NASA Space Grant Fellowship in Science and Engineering. Based on what she has learned from this project, she proposed research to develop multiplex test strips to detect several kinds of pesticides simultaneously with advantages of this method, such as rapid detection in agricultural fields and cost-effectiveness of peroxidase-like nanoparticles. With this fellowship, she attended the AIChE 2019 annual meeting. In 2020, she presented her results at the virtual AIChE 2020 annual meeting. She received the women in chemical engineering (WIC) travel award and an honorable mentioned student award at the student competition in the environmental sensors session. Xiaofan Ruan is a Ph.D. student in the School of Mechanical and Materials Engineering. He studies nanomaterials and devices for biosensing with the interdisciplinary application of 3D-printing for food safety and environmental applications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two PhD students (Eunice Kwon and Xiaofan Ruan) have been trained on this project. Both learned about nanomaterials synthesis, characterization, and signal amplification mechanism. They understood immunoassay principles, mechanisms behind, and techniques for developing lateral flow test strips, along with optimization procedures, determination of the analytical linear range, LOD, and recovery. They came to learn new 3D printing technology and designed multiplexed electrochemical biosensing devices and applied them for simultaneous detection of multiple pesticides. To validate our device using real samples, the students learned food sample processing called the QuEChERS technique (Quick, Easy, Cheap, Effective, Rugged, and Safe). This technique is widely used for pesticide residues analysis in food matrices, including fruits and vegetables. Bi-weekly project meetings served to train them on how to organize their results and improve presentation skills. One of the graduate students presented her results at the virtual AIChE 2020 Annual Meeting. At the AIChE 2020 meeting, she received the women in chemical engineering (WIC) travel award and an honorable mentioned student award at the student competition in the environmental sensors session. How have the results been disseminated to communities of interest?Students will present their results at the Graduate and Professional Students Association (GPSA) Academic Showcase Research Exposition at WSU. One paper was published on Analytica Chimica Acta under the title "Mesoporous Pd@Pt nanoparticle-linked immunosorbent assay for detection of atrazine." One paper was submitted to Biosensors and Bioelectronics under the title "Nanomaterial-enhanced electrochemical immunosensor for simultaneous detection of atrazine and acetochlor on a 3D-printed platform." One review article under the title "Immunoassay technologies for the simultaneous analysis of multiple pesticide residues in food and water" is in preparation and will be submitted to Food Chemistry. Data of HPLC results are collected and will be submitted to Food Chemistry. A PhD student presented her results at the AIChE 2019 Annual Meeting in Orlando, FL, and at the virtual AIChE 2020 Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?Next year, we will focus on validating our biosensing approach with HPLC analysis in real samples. We will evaluate the selectivity, sensitivity, and repeatability of the multiplexed sensor for simultaneous detection of multiple pesticides in juices, fruits, vegetables, and groundwater. The results will be compared with HPLC results in standard solution, spikes samples, and raw samples.
Impacts
What was accomplished under these goals?
During the third year of this project, we developed a multiplexed immunosensor device for simultaneous detection of three pesticides under Aim 2 and explored the feasibility of our technology to detect pesticides in fruits, vegetables, and groundwater under Aim 3. (1) Development of windmill-like multiplexed immunosensor. A windmill-like three-channel lateral flow strip was designed and developed for simultaneous detection of three pesticides, atrazine, acetochlor, and chlorpyrifos. The purpose of this design is to avoid/minimize mutual interference for simultaneous measurements. In the design, the three test strips in the channels shared one circular sample pad (Ø = 10 mm), and each test strip was constructed by one 15 mm × 4 mm absorbent pad, one 25 mm × 4 mm nitrocellulose membrane, and one 10 mm × 4 mm conjugate pad. Other sensor platform parts were 3D printed accessories, including a strip cutter, a strip holder, and an electrode holder. Three screen-printed electrodes were connected and integrated with the test zones of strip channels. The measurement was based on competitive immunoassays. Before testing, the antibodies of atrazine, acetochlor, and chlorpyrifos were conjugated with mesoporous core-shell palladium-@-platinum nanoparticles (Pd@Pt NPs) and coated on the conjugation pad. The three test zones of the test strips were prepared by dispensing BSA-atrazine, BSA-acetochlor, and BSA-chlorpyrifos, respectively, and allowed to dry overnight at room temperature. For the immunoassays, 250 μL of herbicide mixture solution was applied onto the sample pad, and migrated toward the absorbent pads of the three channels driven by the capillary forces. The targets of atrazine, acetochlor, and chlorpyrifos competed with BSA-haptens on the conjugation zones respectively to bind to the limited sites of corresponding antibody-Pd@Pt NPs conjugates coated on the conjugate pads. A time frame of 10 min was optimized for the lateral flow immunoassay. The concentrations of herbicides were determined by the antibody-Pd@Pt NPs on the test lines via their catalytic activity in the redox reaction of thionin and hydrogen peroxide. The test zones of the strips were snipped by the cutter and dropped into the reaction cells sealed above the screen-printed electrodes, which comprises a 100 μL liquid drop formed 1.0 mM thionin acetate and 50 mM hydrogen peroxide. After incubation for 2 mins, the corresponding redox reaction was monitored by differential pulse voltammetry scanning from -0.1 to -0.7 V, with a scanning amplitude of 0.02 V and a pulse width of 0.05 s. (2) Simultaneous detection of atrazine, acetochlor, and chlorpyrifos using the immunosensor. The windmill-like three-channel biosensor was optimized through the pretreatment of conjugate pads by 2% BSA + 2% sucrose in PBS for the anti-atrazine antibody complex, 2% BSA + 2% sucrose + 0.3% Tween-20 in PBS for the anti-acetochlor antibody complex, and by 2% BSA + 2% sucrose in PBS for the anti-chlorpyrifos antibody complex. The concentration of antibody-Pd@Pt NPs applied to the conjugate pad was optimized to be 0.6 mg/mL for the anti-atrazine antibody complex, 0.8 mg/mL for the anti-acetochlor antibody complex, and 0.8 mg/mL for the anti-chlorpyrifos antibody complex. With the optimized sensor platform, simultaneous detection was achieved with the detection limit of 0.22 ng/mL for atrazine, 3.1 ng/mL for acetochlor, and 0.017 ng/mL for chlorpyrifos. (3) Detection of atrazine and acetochlor in real samples with HPLC. HPLC analysis was carried out using a Shimadzu HPLC LC-2030 with a UV detector. The column used was a C18 (4.6 X 250 mm, 5 μm) from Agilent ZOBRAX Eclipse XDB. The mobile phase used methanol-H2O (75-25%, v/v) with the H2O in this mixture adjusted to pH 3 by adding 85% phosphoric acid. With HPLC analysis, LODs were obtained at 10 ng/mL for atrazine and 50 ng/mL for acetochlor. a) Sample preparation (QuEChERS methods) Sample preparation used QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method with modifications to adjust to our lab conditions. The apple juice, watermelon juice, fruit, and vegetable samples (apple, strawberry, green cabbage, and corn) were purchased from the local market. The sample preparation of juice samples was followed by this procedure. First, 30 ml of a juice sample was centrifuged at 8000 rpm for 15 min and filtrated with a syringe filter. NaCl (6 g) was added to 20 ml of the centrifuged and filtrated sample and mixed for 1 min. Then, 20 ml of acetonitrile containing 1% acetic acid (v/v) was added to the sample and mixed for 1 min. Next, the sample was held at room temperature for 1 h, followed by centrifugation at 8000 rpm for 5 min. The upper phase (acetonitrile phase) was transferred to a glass vial and evaporated with a nitrogen evaporator. The evaporated samples were then dissolved into 2 ml of methanol to remove the salt. Finally, two herbicides, atrazine, and acetochlor were spiked into the final solution, and the mixture was filtrated with a syringe filter for running HPLC. b) Detection of atrazine and acetochlor spiked in juice samples Apple and watermelon juice samples were spiked with these two herbicides at four different concentrations: atrazine at 30, 50, 80, and 100 ng/mL and acetochlor at 80, 100, 150, 200 ng/mL, respectively. The presence of the herbicides in the samples was detected by HPLC with the recoveries of 95.4 - 102% (in triplicate). c) Detection of atrazine and acetochlor spiked in real samples Fruit and vegetable samples were spiked with these two herbicides at four different concentrations, same as juice samples: atrazine at 30, 50, 80, and 100 ng/mL and acetochlor at 80, 100, 150, 200 ng/mL, respectively. HPLC analysis resulted in recoveries between 87.7 and 120%.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Analytica Chimica Acta 1116 (2020) 36-44
|
Progress 04/01/19 to 03/31/20
Outputs
Target Audience:PD, Dr. Dan Du, is supervising the PhD student, Xiaofan Ruan and co-supervising PhD student Eunice Kwon. She was invited to give a lecture on "Nanobiosensors for food safety and environmental application" for graduates in Food Science in 2019. She covers nanoscale-based sensing mechanisms, signal amplification schemes and smart sensors for reliable and cost-effective detection of pathogens and pesticides in food. Co-PD, Prof. Bernie Van Wie, is supervising the efforts of PhD student Eunice Kwon and co-supervising the efforts of PhD student, Xiaofan Ruan whose major advisor is Dr. Du. Typically, Van Wie gives feedback at twice monthly team meetings to Kwon and Ruan and has been interacting with Kwon on her first publication, NASA grant application and poster presentation. Co-PD, Prof. Yuehe Lin, is teaching a graduate class on Nanoscience and Nanotechnology in the spring semester 2019 and he includes nanotechnology for agricultural and food systems. He covers nanomaterials synthesis, characterization, nanosensor mechanisms and applications in detection of pathogens, insects, diseases, chemicals, and contaminants in food, water and the agricultural production environment. Eunice Kwon, PhD student, received a NASA Space Grant Fellowship in Science and Engineering. Based on what she has learned from this project, she proposed research to develop multiplex test strips to detect several kinds pesticides or herbicides simultaneously with advantages of this method such being rapid detection in agricultural fields and cost-effectiveness due to use of peroxidase-like nanoparticles. With this fellowship, she attended the annual AIChE conference. Xiaofan Ruan is a Ph.D. student in the School of Mechanical and Materials Engineering. He is studying nanomaterials and devices for biosensing with interdisciplinary application of 3D-printing for food safety and environmental applications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two PhD students (Eunice Kwon, and Xiaofan Ruan) have been trained on this project. Both learned about nanomaterials synthesis, characterization and signal amplification mechanism. They started to understand immunoassay principles, mechanisms behind and technique for developing lateral flow strips, along with optimization procedures, determination of the analytical linear range, LOD and recovery. They came to lean new 3D printing technology and designed two-channel and three-channel immunochromatographic electrochemical biosensing devices and applied them for simultaneous detection of multiple pesticides. To validate our device using real samples, the students leaned food samples processing called as QuEChERS technique (Quick, Easy, Cheap, Effective, Rugged and Safe). This technique is widely used for pesticide residues analysis in food matrices including fruits and vegetables. Bi-weekly project meetings served to train them on how to organize their results and improve presentation skills. A presentation was made by one of the graduate students at the 2019 Annual American Institute of Chemical Engineers (AIChE) Conference in Orlando and a few students expressed interest in getting involved in the project. A visiting scholar from China joined the team in April 2019 for a yearlong exchange. How have the results been disseminated to communities of interest?Students will present their results at the Graduate and Professional Students Association (GPSA) Academic Showcase Research Exposition at WSU. ELISA related result has been submitted and revised for publication on Analytica Chimica Acta under the title "Mesoporous Pd@Pt Nanoparticle-linked Immunosorbent Assay for Detection of Atrazine". Multiplex biosensor results are collected and will be submitted soon under the title "Nanomaterial-enhanced electrochemical immunosensor for simultaneous detection of atrazine and acetochlor on a 3D-printed platform". Data of lateral flow strip results are collected and will be submitted to Biosensors & Bioelectronics. A PhD student presented her results at the 2019 Annual AIChE Conference in Orlando, FL. What do you plan to do during the next reporting period to accomplish the goals?Next year, we will focus on refinement of the multiplex immunosensing device and validate our biosensing approach with HPLC in real samples. We will evaluate the selectivity, sensitivity and repeatability of the multiplex sensor for simultaneous detection of multiple pesticides in juices, fruits, vegetables and groundwater. The results will be compared with HPLC results in standard solution, spikes samples and raw samples.
Impacts
What was accomplished under these goals?
During the second year of this project, we focused on measurements of multiple pesticides and development of multiplex immunosensor device under Aim 2 and start to explore the feasibility of our technology for detection of pesticides in fruits, vegetables and groundwater under Aim 3. (1) Synthesis and characterization of peroxidase-like Pd@Pt nanoparticles (NPs). Mesoporous Pd@Pt nanoparticles were synthesized and characterized by transmission electron microscopy (TEM). To synthesize Pd@Pt NPs, 25 mM K2PtCl4 was mixed with 20 mM Na2PdCl4 and HCl. Then 20 mg of Pluronic® F127 was dissolved into the mixture and 2 mL ascorbic acid (AA) was added. The mixture was placed into a 35 °C water bath for 4 h. During this time, NPs formed as indicated by a color change from brown to black. In the presence of an AA reducing agent, PdCl42- reduction occurs at a higher rate than that of PtCl42- and therefore a NP forms where Pd is the dominate species in the core. This results in creation of a Pd-rich seed template within the spherical micelles and the metal seed is stabilized by the interaction between the hydrophobic polypropylene oxide group of the surfactant and the metal surface. Then the amphiphilic surfactant adsorbed on the surface of the metal seed promotes binding of PtCl42- ions due to the presence of the hydrophilic polyethylene oxide group and further reduction to Pt leads to its deposition around the Pd core. With the surfactant as a structure-directing agent, Pt shell overgrowth on the Pd seed. From TEM images, mesoporous Pd@Pt NPs demonstrated they consist of a branched structure, but are nearly spherical, consistent in structure and size (~50 nm) and have very high surface areas as supported by the observable fractal-patterned structure. Also, literature shows that Pd dominates in the inner core while Pt is more highly concentrated in the outer shell, especially in the spiked ends protruding into the solution. Pd@Pt NPs with strong peroxidase-like activity were observed in its ability to catalyze the H2O2-,3',5,5'-tetramethylbenzidine (TMB) redox reaction while maintaining stability over a 1-11 pH range and 4-90 °C temperature range. (2) Optimization and Development of Pd@Pt NPs-enhanced lateral flow immunoassay for detecting atrazine. The optimization studies of lateral flow immunoassay for detection of atrazine detection was performed including (1) blocking buffer on the conjugate pad to prevent non-specific binding, (2) pH of antibody and NPs conjugation (Ab-NP conjugates), and (3) the concentration of Ab-NPs on the conjugate pad. Since the intensity of test line at the 2% bovine serum albumin (BSA) and 3% sucrose in phosphate-buffered saline with Tween-20 is higher than that of other buffers such as 1% BSA and 3% sucrose buffer, 3% sucrose buffer and 1% BSA buffer, the 2% BSA and 3% sucrose buffer was chosen as the blocking buffer. The pH of Ab-NP conjugates was optimized at pH 8 - 8.3 because test line intensities at pH 8 and 8.3 were higher than those of other pH. The optimal amount of Ab-NP conjugates was determined at 0.5 mg/mL due to higher difference intensity depending on the presence of atrazine in sample than that of others. The optimized conditions of 2% BSA and 3% sucrose blocking buffer with 0.5 mg/mL of Ab-NP conjugates at pH 8 - 8.3 were used to detect different concentration of atrazine from 10 ng/mL to 500 ng/mL. The intensity of test line decreases with increasing concentration of atrazine because the lateral flow immunoassay for atrazine detection follows the competitive immunoassay. ?(3) Development of multiplex immunosensor device. A two-channel lateral flow strip was designed and developed for simultaneous detection of multiple pesticides. The strips were constructed to have a sample pad, conjugation pad, nitrocellulose membrane and absorbent pad on a supporting membrane card. Each channel of a strip contained a 12 mm x 4 mm absorbent pad, a triangular sample pad that allowed sample to exit simultaneously out the broad base of an isosceles triangle to one of two test strips. The control line on both channels were created by dispensing goat-anti-mouse IgG antibody. The test lines were prepared by dispensing BSA-atrazine and BSA-acetochlor respectively and allowed to dry overnight at room temperature. Finally, the two types of antibody-Pd@Pt conjugates, one for atrazine and one for acetochlor, were coated onto the conjugation pad to finish the fabrication of lateral flow strip. Assay parameters of the two-channel lateral flow strip were optimized including pretreatment of conjugate pads and concentration of coated antibody-nanoparticle complex. The optimal pre-treatment of the conjugate pads was found to be 2 % BSA + 2 % sucrose in PBS for the anti-atrazine antibody complex ([anti-atrazine]-Pd@Pt NPs); and the 2 % BSA + 2 % sucrose + 0.3 % Tween-20 in PBS for the anti-acetochlor complex ([anti-acetochlor]-Pd@Pt NPs), respectively. The optimal concentration of coated antibody-Pd@Pt NPs conjugates was determined to be 0.6 mg/mL for the anti-atrazine antibody complex and 0.8 mg/mL for the anti-acetochlor antibody complex. The amount of antibody-Pd@Pt NPs on the test lines was determined via their overall catalytic activity in the electrochemical redox reaction between thionin acetate (1mM) and hydrogen peroxide (50 mM) in a 3d-printed device. The corresponding redox reaction is monitored by differential pulse voltammetry (DPV) scanning from -0.8 to -0.1 V, with a scanning amplitude of 0.05 V and pulse width of 0.05 s. By using the optimized multiplex immunosensor device, the simultaneous detection of atrazine and acetochlor was realized with a limit of detection of 0.3 ng/mL and 3.3 ng/mL, respectively. To evaluate the feasibility, the device was employed for detecting spiked samples containing atrazine and acetochlor residues, and an overall recovery in the 90.8% to 117 % range was obtained. (4) Evaluation of immunosensor device with with HPLC. To detect atrazine and acetochlor in spiked and/or real samples with HPLC analysis, the standard herbicide samples were studied to yield standard curve. HPLC analysis was carried out using a Shimazhu HPLC LC-2030 with UV detector. The column was used C18 (4.6 X 250 mm, 5 μm) from Agilent ZOBRAX Eclipse XDB. Mobile phase was used methanol-H2O (75-25 v/v) and pH of H2O was adjusted to pH 3 by 85% phosphoric acid. The limit of detections (LODs) were detected as 10 ng/mL for atrazine and 50 ng/mL for acetochlor.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Biosensors and Bioelectronics, 2019, 142, 111498
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Small, 2019, 1901485
- Type:
Websites
Status:
Published
Year Published:
2019
Citation:
https://news.wsu.edu/2019/10/28/new-research-makes-potent-artificial-enzymes-single-atom-architecture/
|
Progress 04/01/18 to 03/31/19
Outputs
Target Audience:Co-PD, Prof. Yuehe Lin, is teaching a graduate class on Nanoscience and Nanotechnology in the spring semester 2019 and he includes nanotechnology for agricultural and food systems. He covers nanomaterials synthesis, characterization, nanosensor mechanisms and applications in detection of pathogens, insects, diseases, chemicals, and contaminants in food, water and the agricultural production environment. Co-PD, Prof. Bernie Van Wie, is supervising the efforts of PhD student Eunice Kwon and co-supervising the efforts of PhD student, Xiaofan Ruan whose major advisor is Dr. Du. Typically, Van Wie gives feedback at twice monthly team meetings to Kwon and Ruan and has been interacting with Kwon on her first publication, NASA grant application and poster presentation. PD, Dr. Dan Du, was invited to give a lecture on "Nanobiosensors for food safety and environmental application" for graduates in Food Science in 2018. She covers nanoscale-based sensing mechanisms, signal amplification schemes and smart sensors for reliable and cost-effective detection of pathogens and pesticides in food. Eunice Kwon, a PhD student, received a NASA Space Grant Fellowship in Science and Engineering. Based on what she has learned from this project, she proposed research to develop multiplex test strips to detect several kinds pesticides or herbicides simultaneously with advantages of this method such being rapid detection in agricultural fields and cost-effectiveness due to use of peroxidase-like nanoparticles. With this fellowship, she plans to attend the annual AIChE conference. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two PhD students are working on this project. Both of them learned about nanomaterials synthesis, characterization and signal amplification schemes. They came to understand principles behind ELISAs, mechanisms behind and technique for developing lateral flow strips for immunoassays, along with optimization procedures, determination of the analytical linear range, LOD and recovery. Based on their results, the PhD students have had a chance to attend the Washington State University (WSU) Academic Showcase where well over a hundred posters are presented by graduate students and assessed by a panel of judges with prizes awarded. Bi-weekly project meetings served to train them on how to organize their results and improve presentation skills. A presentation was made by one of the graduate students at an undergraduate research symposium and a few students expressed interest in getting involved in the project. How have the results been disseminated to communities of interest?Students will present their results at the Graduate and Professional Students Association (GPSA) Academic Showcase Research Exposition at WSU. ELISA related results will be submitted soon for publication under the title "Pd@Pt nanoparticle-amplified enzyme-linked immunosorbent assay for detection of atrazine". Data are being collected for a second paper on lateral flow strip results. A PhD student also plans to present her results at the 2019 Annual American Institute of Chemical Engineers (AIChE) Conference in Orlando, FL. What do you plan to do during the next reporting period to accomplish the goals?Next year, we will focus on development and refinement of the multiplex immunosensing device and establish a nanoparticle electrochemical detection approach. We will evaluate the selectivity and sensitivity of the multiplex sensor for simultaneous detection of multiple pesticides. Optical and electrochemical detection will be performed in parallel and compared.
Impacts
What was accomplished under these goals?
During the first year of this project, we focused on synthesis and characterization of Pt-based nanomaterials under Aim 1 and started work on a multiplex immunosensor device under Aim 2. (1) Two kinds of Pt-based nanomaterials, mesoporous Pd@Pt nanoparticles and 2D Pt-Ni(OH)2 nanosheets were synthesized. Transmission electron microscopy (TEM), X-ray diffraction (XRD) and energy dispersive spectroscopy (EDS) have been used for characterization of the morphologies of the nanomaterials. We observed that Pd@Pt nanoparticles showed a nearly spherical uniform and mesoporous core-shell structure with an average particle size of ~50 nm. The TEM images of Pt-Ni(OH)2 exhibited uniform distribution of Pt nanoclusters on Ni(OH)2 nanosheets. The size distribution of Pt nanoclusters on the nanosheets is 2 nm. EDS studies for analyzing the Pt-Ni(OH)2 composition displayed both Pt and Ni peaks, evidence of the formation of Pt nanocrystals on Ni(OH)2 nanosheets. The catalytic activities of Pd@Pt nanoparticles and Pt-Ni(OH)2 nanosheets were further conducted and compared with horseradish peroxidase (HRP), an enzyme that catalyzes the reaction of a non-colored to colored substrate for spectrophotometric detection. Both nanomaterials displayed high peroxidase-like activity. (2) A series of detection optimizations were performed, including antibody-nanomaterials conjugation (Ab-N), incubation time, blocking buffer selection and pre-treatment of paper strips. We observed that signal increased with the increase in concentration of Ab-N to reaching a steady value in the 0.5 to 1.0 mg/ml range. Considering the high cost of antibodies, 0.5 mg/ml conjugates were selected. For Enzyme-Linked Immunosorbent Assays (ELISAs), a time of 40 min was used for conversion to a colored substrate since a longer time could result in a large nonspecific signal. For test strips, a time frame of 10 min was enough to produce sufficient signal. Minimization of nonspecific adsorption was achieved at 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Higher BSA concentrations resulted in decreased signal strength which may be due to steric hindrance of the large Ab-N species which need to reach the surface where BSA-herbicide conjugates are bound. In lateral flow immunoassays, pre-treatment of the test strip is an important step. Based on maximizing cyclic voltammetry signals, we found that a 2% BSA / and 2% sucrose buffer containing 0.3% Tween 20 is the best solution tested for treating the test strip before immobilization of Ab-N. (3) A two channel lateral flow strip was designed and developed for simultaneous detection of multiple pesticides. The strips were constructed to have a sample pad, conjugation pad, nitrocellulose membrane and absorbent pad on a supporting membrane card. Each channel of a strip contained a 12 mm x 4 mm absorbent pad, a 25 mm x 4 mm nitrocellulose membrane, a 10 mm x 4 mm conjugation pad and a 25 mm x 9 mm large triangular sample pad that allowed sample to exit simultaneously out the broad base of an isosceles triangle to one of two test strips. The control line on both channels were created by dispensing goat-anti-mouse IgG antibody. The test lines were prepared by dispensing BSA-atrazine and BSA-acetochlor respectively, and allowed to dry overnight at room temperature. Finally, the two types of antibody-Pd@Pt conjugates, one for atrazine and one for acetochlor, were coated onto the conjugation pad to finish the fabrication of lateral flow strip. (4) Mesoporous Pd@Pt nanoparticles-amplified ELISAs were established and optimized for detection of the herbicide atrazine. Due to the strong peroxidase-like catalytic activity of Pd@Pt nanoparticles, we are able to supply stable, low cost and sensitive nanomaterials conjugated to antibodies. The limit of detection (LOD) for atrazine is 0.5 ng/mL which is comparable or better than conventional methods, yet without the cost, purification and stability issues associated the use of peroxidase enzymes. We applied the technique to detection of spiked well and pond water samples and found recoveries between 88 and 118%, which is acceptable for ELISA approaches. In using lateral flow strips, the detection limit is 0.1 ng/mL, and the recoveries for spiked water samples containing atrazine between 90.8 and 117 %.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Pt-Ni(OH)2 Nanosheets Amplified Two-way Lateral Flow Immunoassays with Smartphone Readout for Quantification of Pesticides
|
|