A Nanozyme- and Ambient Light-Based Smartphone Platform for

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A Nanozyme- and Ambient Light-Based Smartphone Platform for Simultaneous Detection of Dual Biomarkers from Exposure to Organophosphorus Pesticides Yuting Zhao, Mingming Yang, Qiangqiang Fu, Hui Ouyang, Wei Wen, Yang Song, Chengzhou Zhu, Yuehe Lin, and Dan Du Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00837 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Analytical Chemistry

A Nanozyme- and Ambient Light-Based Smartphone Platform for Simultaneous Detection of Dual Biomarkers from Exposure to Organophosphorus Pesticides Yuting Zhaoa,b,1, Mingming Yanga,1, Qiangqiang Fua, Hui Ouyanga, Wei Wena, Yang Songa, Chengzhou Zhua, Yuehe Lina*, Dan Dua* a

School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164, United States Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, People's Republic of China KEYWORDS: Organophosphorus; Butyrylcholinesterase monoclinal antibody; Smartphone; Transparent immunochromatographic test strips; Polyphenol oxidase-like nanozyme b

ABSTRACT: A transparent, lateral-flow test strip coupled with a smartphone-based ambient light sensor was first proposed for detecting enzymatic inhibition and phosphorylation. The principle of the platform is based on the simultaneous measurement of the total amount of the enzyme and enzyme activity to biomonitor exposure to organophosphorus (OP) pesticides. In this study, butyrylcholinesterase (BChE) was adopted as the model enzyme and ethyl paraoxon was chosen as an analyte representing OP pesticides. The total amount of BChE was quantified by a sensitive colorimetric signal originating from a sandwich immunochromatographic assay utilizing PtPd nanoparticles as a polyphenol oxidase-mimicking probe, which provided a higher sensitivity than that of a peroxidase-like probe. In the sandwich immunoassay, only one antibody against BChE was simultaneously utilized as the recognition antibody and the labelling antibody due to the tetrameric structure of native BChE. The BChE activity was estimated by another colorimetric signal using the Ellman assay. Both colorimetric signals on two separated test strips were detected by the smart phone-based ambient light sensor. The proposed sensor operated with an LED in a 3D-printed substrate, which emitted excitation light and transmitted it through a transparent, lateral-flow test strip. With the increase in the colorimetric signal in the test line of the test strip, the intensity of the transmitted light decreased. The smartphone-based sensor showed excellent linear responses for assaying the total amount of BChE and active BChE ranging from 0.05 to 6.4 nM and 0.1 to 6.4 nM, respectively. A high portability and low detection limit were simultaneously realized in the common smartphone-based device. This low-cost, portable, easy-operation and sensitive immunoassay strategy shows great potential for on-line detection of OP exposure and monitoring other disease biomarkers.

The extensive use of organophosphorus (OP) compounds, including pesticides and chemical nerve agents, results in an increasing risk to human health.1-3 Therefore, there is an urgent need to develop low-cost, rapid, sensitive methods for the detection of OP compounds. At present, the most commonly used biomarkers for OP include unbound, free OP compounds, their hydrolysis metabolites and phosphorylated enzyme adducts (OP-ChE).4-8 There have been many sophisticated instrument-based analytical methods reported for assaying free OP compounds and their metabolites. However, due to the requirement for large instruments, complicated sample treatment, skilled personnel and high assay cost, these methods are not suitable for rapid testing and field assays. Due to their easy operation, high specificity, high-throughput and high sensitivity, nanoparticle-based immunoassays for OP adducts can be adopted as potential approaches to monitor exposure to organophosphorus (OP) pesticides.9-11 For these immunoassays, two different antibodies are needed to conduct sandwich-type protocols for biomonitoring OP adducts. Therefore, there is significant motivation to design a sandwich-type immunoassay that uses only one antibody. Owing to their ultra-facile operation and short assay time, immunochromatographic test strips (ICTS) have been applied

widely for point-of-care testing in human healthcare.12-15 Recently, colloidal nanoparticles, especially gold nanoparticles, have been utilized as the colorimetric probe for fabricating ICTS, such as the home pregnancy HCG test strip, HIV test strip, HBsAg test strip and so on.16,17 However, drawbacks such as a low accuracy and sensitivity have restricted the further applications of OP adducts for detection of trace concentrations. Currently, novel optical or electrochemical nanoparticle-based ICTS with lower detection limits have been proposed, which provide a new pathway for quantifying OP adducts with a high sensitivity.18-20 However, disadvantages, such as the requirement of sophisticated instruments and complicated assay procedures, lead to high assay costs and long assay times. It would be possible to address this issue by means of facile sensors using smartphones as the detection instruments. There were over 2 billion smartphone users around the world in 2016, and the number of users is estimated to reach 6.1 billion in 2020, representing 70 percent of the global population.21 Utilized as analytical platforms, smartphones show remarkable portability for point-of-care testing of different analytes.22 Moreover, many smartphones are equipped with high-performance cameras, USB ports, Bluetooth, and ambi-

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ent light sensors (ALS), which are potential analysis terminals for conducting numerous optical sensors. Due to the universality and multifunctional properties of smartphones, researchers have been actively engaged in combining smartphones and ICTS to develop novel optical camera-based sensors.23-26 Using a smartphone camera to collect colorimetric signals, Martina et al. developed a chemiluminescent ICTS method to quantitatively detect salivary cortisol at trace concentrations.27 Similarly, Lee et al. presented a silver-enhanced gold nanoparticle-based ICTS for rapid quantification of vitamin D using a smartphone.28 You et al. conducted a highly sensitive fluorescent ICTS for two heart failure biomarkers using a smartphone as a fluorescence reader.29 Yeo et al. proposed a smartphonebased fluorescent ICTS with an efficient reflective light collection module for three different avian influenza subtypes.30 These above smartphone-based ICTS mostly rely on utilizing correlative APPs to analyze the colorimetric intensity of the photos of the test lines in ICTS. However, there are some drawbacks to this method: (1) photos taken with a camera are easily distorted and do not correspond well with the actual signals of the test lines in ICTS; (2) at present, the principle of smartphone-based assay analysis is mainly based on grayscale scanning or RGB analysis, so the obtained results are equivalent to secondary data; thus, the accuracy is limited; (3) there is a dramatic difference in the photo resolution among different smartphones, and thus, the reproducibility of photo analysis-based optical ICTS developed on different smartphones cannot meet the demand of point-of-care testing; and (4) in the analysis process, precise optical accessories, such as filters, gratings and prisms, are needed for high photo resolutions and high analysis sensitivity, which conversely makes smartphone-based sensors complicated and expensive. In contrast, ambient light sensors (ALS) have been seldom utilized in smartphone-based biosensors12. In the ALS-based sensor, the intensity of the optical beam is attenuated due to absorption by the medium; meanwhile, the absorption of the medium changes with differences in the analyte concentration. Superior to camera-based sensors, the optical signal is the primary data, resulting in a higher sensitivity and precision in ALS-based sensors. To improve the practicality of ALS-based sensors, it is vital to adopt a colorimetric reaction with a high sensitivity. Enzymatic browning is a ubiquitous phenomenon among post-harvest fruits and vegetables. Fruits and vegetables contain abundant polyphenol oxidase(PPO) and peroxidase(POD), which oxidizes natural phenols to quinones in the presence of oxygen. Eventually, these quinones polymerize to form dark brown pigments (melanosis) on the surface of the food.31,32 Furthermore, the oxidation and polymerization reactions catalyzed by polyphenol oxidase are extremely rapid and potentially effective coloration reactions for ambient light sensors. Inspired by this natural phenomenon, PtPd NPs which exhibited excellent catalytic activity for phenols were prepared as colorimetric probes for conducting an ALS-based ICTS for the first time. We first developed an ALS-based ICTS with a high sensitivity, high portability, and low cost to detect both butyrylcholinesterase (BChE) activity and the total amount of BChE (including the enzyme inhibited by OP and the active enzyme) simultaneously. In the proposed ICTS, as the colorimetric signal in the test line of test strip increases, the intensity of the

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transmitted light decreases. Due to the tetrameric structure of BChE, only one monoclonal antibody (MAb) against BChE was necessary to simultaneously function as both the capture antibody and detection antibody. 33-37 Because a sandwich type was adopted, the total amount of BChE was detected based on the catalytic activity of the PtPd NPs, which oxidized catechol to form a dark-brown product to attenuate the light intensity in the ALS-based ICTS. The BChE activity was also evaluated by means of an Ellman assay based on the ICTS. The proposed ICTS showed excellent linear responses for assaying the total amount of BChE and active BChE ranging from 0.05 to 6.4 nM and 0.1 to 6.4 nM, respectively. Owing to the remarkable portability, low cost, short assay time and high sensitivity, the novel ALS-based ICTS showed great potential in various areas such as clinical diagnosis, environmental monitoring and food safety. EXPERIMENTAL SECTION Materials and Reagents. Human butyrylcholinesterase (ab96367) (>100U), BChE monoclonal antibody (BChE MAb) (ab17246), goat anti-mouse IgG and HRP-conjugated goat anti-mouse IgG (HRP-Ab2) were all purchased from Abcam Inc. (USA). Ethyl paraoxon was purchased from Chem Service, Inc. (USA). Butyrylthiocholine chloride (BTCh), 5,5’dithiobis (2-nitrobenzoic acid) (DTNB), phosphate buffer saline (PBS), Tween-20, 3,3’,5,5’-Tetramethylbenzidine (TMB), Pluronic F127, K2PtCl4 (Pt, 44.99%), Na2PdCl4 (Pd, 49.98%), hydrochloric acid (HCl, 39%), sodium hydroxide (NaOH), ascorbic acid (AA) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (USA). 40 nm gold nanoparticles (Au NPs) was purchased from nanoComposix (USA). bicinchoninic acid assay (BCA) kit were purchased from Thermo Scientific (Rockford, IL). Microplates for enzymelinked immunosorbent assay (ELISA) were purchased from Becton (USA). Fiber conjugate pad, fiber sample pad, nitrocellulose membrane and absorbent pad were obtained from Millipore (USA). Ultrapure water from a Millipore Milli-Q water purification system was used for experiments. LEDs were obtained from Shenzhen Octai Co., Ltd. The Battery Lithium AA Size 1.5 Volt was obtained from Washington State University store. Transparent plastic scale board, resistances, switches, and wires were purchased from a local store. Apparatus. The separation of PtPd NPs-conjugated BChE MAb (PtPd NPs- BChE MAb) was performed on a centrifuge (Eppendorf 5417C; Eppendorf, USA). The test strip was fabricated using a Guillotine Cutting System (Bi oDot LTD; USA). The BChE MAb was dispensed on the nitrocellulose membrane by using BioDot BioJet BJQ 3000


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Fig. 1. Illustration of the principle of the simultaneously immunodetection of the BChE activity and total amount of BChE (A). PtPd NPs for signal amplification (B). Smartphone-based ALS (C). Fabrication of ICTS. As seen in Fig. 1A, the ICTS consisted of a sample pad, a conjugated pad, a nitrocellulose memdispenser (Irvine, CA). The absorbance assays were carried brane and an absorbent pad. Prior to use, the sample pad and out on a Tecan Safire2 microplate reader (Tecan, Switzerland). conjugated pad were pretreated with 10 mM pH 7.4 PBS conThe transmission electron microscopy (TEM) images were taining 0.05% Tween and 1% BSA and then dried at 37 °C for obtained with a Philips CM200UT instrument. Smartphone 2 hours. BChE MAb (1mg/mL) was dispensed on the nitrocel(Huawei Honor 6; China). The ALS device was print on 3D lulose membrane at the rate of 1 µL/cm to form the test line Printer (Hangzhou Shining 3D Tech Co., Ltd; China). and then dried at room temperature overnight. Finally, the four Preparation of PtPd NPs. PtPd NPs were synthesized by sections were assembled on a transparent plastic scale board, using a previous method with a little modification.38 Briefly, cut into 4 mm strips and stored at room temperature. Pluronic F127 (20 mg) was dissolved in mixing solution conDesign of the ALS-based device. Our ALS-based device taining K2PtCl4 (1.8 mL, 20 mM), Na2PdCl4 (0.2 ml, 20 mM) for analysis was implemented on an Android phone (Huawei and HCl (44 µL, 6 M). After adding the reducing agent AA Honor 6). The device was a 3D-printed holder containing an (2.0 mL, 100 mM), the mixture was continuously sonicated in LED as a light source, a button cell, a resistor for regulating a 35 ℃ water bath for 4 h. The final product was centrifuged the intensity of the excitation light and a slot for fixing the test and washed with acetone and water for five times, respectively. ICTS. The blue LED was selected as the light source to simulThe final product was dispersed in 5 mL water and stored in taneously quantify the total amount of BChE and the activity 4 ℃ until use. of BChE (Fig. S1, Table S1). The resistance of the resistor was Preparation of PtPd NPs-conjugated BChE MAb (PtPd 10 Ω for optimal light intensity. NPs-BChE MAb). BChE MAb modified PtPd NPs were prePreparation of OP-BChE. 5 µL of 75 mM ethyl paraoxonpared according to our previous method.39 Firstly, the pH of acetone solution (the volume of acetone less 5% of total volthe PtPd nanoparticles solution was adjusted to 8.2-8.5 by ume) was mixed with 100 µL of 1mg/ml BChE, then diluted adding 0.02 M K2CO3. Then, 5 µL of BChE MAb (1 mg/mL) with PBS buffer or human plasma, followed by a 72-hour inwas added into the 1 mL of adjusted Pt-Pd NPs solution. After cubation at room temperature. Meanwhile, BChE solution incubation for 60 min at room temperature, 110 µL of 10.0 without the pretreatment of paraoxon-acetone was used as the wt% BSA was added to the mixture followed by 30 min incucontrol. bation. After that, the mixture was centrifuged at 10,000 rpm Assay procedure for the total amount of BChE. As seen for 8 min and washed twice with 10 mM pH 7.4 PBS containin Fig. 1A, the PtPd NPs- BChE MAb was deposited on the ing 1% BSA. Finally, the resultant PtPd NPs conjugated BChE conjugated pad of the assembled ICTS. 70 µL sample solution MAb was suspended in 100 µL of PBS buffer containing 2% containing BChE and OP-BChE was rapidly added onto the BSA and 3% sucrose. sample pad when the PtPd NPs-BChE MAb was not dried to



ensure the tracer MAb (PtPd NPs-BChE MAb) to migrate with the sample solution. When the mixed solution reached the test line (Fig. 1B (1)), the sandwich-type immunoreaction was complete within 20 min. After that, 2 µL of catechol (0.5 %) and H2O2 (1M) solution was added to the test line for the coloration reaction. One minute later, the transmitted light intensity through the test line was detected with the ALS-based smartphone sensor and recorded by light meter application in the smartphone. Assay procedure for the BChE activity. 70 µL sample solution was added onto the sample pad of another test strip. Driven by capillary force, the solution migrated through the whole strip. In the process, an immunoreaction between BChE (including phosphorylated BChE) and immobilized BChE MAb occurred on the test line (Fig. 1B (2)). After that, an Ellman assay was conducted to evaluate the activity of BChE.40-42 In detail, 2 µL of BTCh (5 mg/mL) and DTNB (0.5 mg/mL) solution was added onto the test line. 5 min later, the colorimetric signal was detected using the ALS-based smartphone sensor and recorded by light meter application in the smartphone. RESULTS AND DISCUSSION Design of the ALS-based device. As seen in Fig. 1C, the proposed ALS-based device was consolidated into a 3Dprinted black cuboid plastic holder (length, 74 mm; width, 50 mm; height, 28 mm) with a gross weight of 60 g. The device included an LED, a resistor and two batteries (1.5 V). The LED was utilized to provide a stable, external emission light source. The resistor was used to adjust light intensity for optimal analysis performance. The battery was used to power the LED. There was a slot (4.1 mm in width) through the bottom of the holder for fixing the ICTS, and a hole (2 mm х 2 mm) in the slot below the LED for sensing the transmitted light intensity. The black holder eliminated the interference from ambient light; therefore, the ALS of the smartphone only received light from the LED in the device. In the sensing process, the ICTS was fixed in the slot, and the test line was adjusted to coincide with the hole. With the increase in the color in the test line, the transmitted light intensity decreased. Lastly, the colorimetric signal of the substrate in the test line was collected through the ALS sensor of the smartphone to evaluate the total amount of BChE and the BChE activity. In detail, the catechol coloration and DTNB coloration were utilized to quantify the total amount of BChE and the BChE activity, respectively. Due to the high catalytic activity of the PtPd NPs in the presence of H2O2, catechol was oxidized and polymerized to form a dark brown product in the test line. The brown intensity increased with the increase in the total amount of BChE because a sandwich model was adopted. Owing to the absorbance effect of the brown product, the transmitted light decreased with the increase in the total amount of BChE. Similarly, the transmitted light intensity decreased with the increase in the BChE activity in the Ellman assay. Principle of the ICTS assay. Natural BChE is a protein with a tetrameric structure bearing four identical antigenic determinants that can simultaneously bind four MAbs. 33-37 Therefore, MAb can be used as both a capturing and labeling antibody because the captured MAb binds to an antigenic determinant of BChE while the labeling antibody binds to another one. In the proposed assay, BChE MAb (ab17246) was adopted as a simultaneous recognition agent and tracer antibody.

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In the ICTS assay, BChE MAb was immobilized as a recognition reagent on the test line to bind BChE and OPBChE. The activity of BChE MAb in recognizing BChE and OP-BChE was tested with an ELISA assay. As seen in Fig. S2, the absorbance values originating from BChE and OP-BChE at the same concentration showed no significant differences, indicating that BChE MAb could bind BChE and OP-BChE simultaneously. To quantify the total amount of BChE, PtPd NPs conjugated to BChE MAb were deposited on the conjugated pad as a colorimetric probe. The sample solution was added onto the sample pad, and then, it migrated through the whole test strip under capillary force. When it reached the conjugated pad, it migrated with the PtPd NP-conjugated BChE MAb to the nitrocellulose membrane. Then, two sandwich immunoreactions occurred between the mixed solution and the immobilized BChE MAb to form BChE MAb/BChE/PtPd NPs-BChE MAb and BChE MAb/OPBChE/PtPd NPs- BChE MAb. After the catechol coloration, the colorimetric signal on the test line was determined by the ALS-based device to quantify the total amount of BChE. Similarly, an Ellman assay for the BChE activity was also conducted using the same procedure without PtPd NP-conjugated BChE MAb on the conjugated pad. The coloration reaction was the BTCh-DTNB system.

Fig. 2. (A) TEM image of PtPd NPs. (B) Zeta potential of PtPd NPs and PtPd-BChE MAb. Characterization of the PtPd NPs and PtPd NPconjugated BChE MAb. Metal precursors, including Pt and Pd, can be reduced simultaneously by AA in the liquid phase to form a faceted crystal morphology. In the synthesis procedure, Pluronic F127 was utilized as a surfactant template to create faceted crystals. Therefore, the resultant bimetallic PtPd NPs could create a high surface area on a concave surface. As seen in Fig. 2A, the size of the prepared PtPd NPs was approximately 30 nm with a uniform snowflake structure. By means of electrical reaction, the PtPd NPs were labelled onto the BChE MAb according to methods described in a previous publication.42 As seen in Fig. 2B, the zeta potential of the PtPd NPs was approximately


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Fig. 3. (A) Photographs of Au NPs (a), PtPd NPs (b), PtPd NPs after adding TMB and H2O2 (c), PtPd NPs after adding catechol and H2O2 (d). The concentration of NPs from up to down was 0, 5, 10, 15, 20, 25, 30 and 35 µg/mL. respectively. Absorbance of different concentration of Au NPs (B), PtPd NPs (C), PtPd NPs after adding TMB and H2O2 (D) and PtPd NPs after adding catechol and H2O2 (E). -44 mV. After conjugation with BChE MAb, the zeta potential of BChE MAb-PtPd NPs was -20 mV. The distinctive change in the zeta potential indicated the labeling process was successful. Additionally, the quantity of the conjugated BChE MAb was determined by a bicinchoninic acid (BCA) protein assay. The results of the BCA assay revealed that approximately 80% of BChE MAb was attached to the PtPd NPs. Optimization of the assay conditions. As key parameters, some assay conditions, including the conditions of the substrate, pH value, volume of the tracer MAb and reaction time were optimized for the ideal analytical performance of the ALS-based ICTS. In the assay for the total amount of BChE, as a nanozyme, PtPd NPs had good catalytic activity for oxidizing TMB and catechol. In the presence of H2O2, the PtPd NPs catalyzed the TMB oxidation, resulting in a deep-blue colored solution within 12 min. Similarly, the PtPd NPs catalyzed H2O2-induced catechol to generate brown complex polymers in a shorter time. Coloration assays were tested to compare the sensitivity of different colorimetric reagents. As seen in Fig. 3Aa and 3B, Au NPs were utilized as the colorimetric reagent, the absorbance remained almost unchanged with increasing its concentration from 0 to 35 µg/mL. As seen in Fig. 3Ab and 3C, the PtPd NPs were adopted as the probe. Without further coloration, the result was similar to the AuNPs. As seen in Fig. 3Ac, 3Ad, 3D and 3E, after the coloration of TMB or catechol, the colorimetric signals increased dramatically with increasing the concentration of PtPd NPs. Compared with the TMB coloration, the catechol coloration showed a higher sensitivity because the absorbance of catechol coloration was higher than the TMB coloration. Additionally, the pH value and volume of tracer MAb were optimized with and without the substrate (TMB and catechol). In the catechol system, as seen in Fig. 4A, the transmitted light

intensity reached a minimum value when the pH was 7.5; for the tracer MAb, the optimal volume was 3 µL because the transmitted light intensity trended to a minimum (Fig. 4B). An excess of MAb did not decrease the transmitted light intensity, probably due to stereohindrance between the BChE/OP-BChE adducts and antibody-modified PtPd NP conjugations. The reaction time after adding substrate was also optimized. Before adding the substrates, the transmitted light intensity is about 6800 lux. The transmitted light intensity reached a minimum value (5803 lux) after adding the substrate for 1 min (Fig. 4C), indicating the reaction rate is much faster than that of the TMB system, which required 12 min to reach the lowest transmitted light intensity. Obviously, 1 min was selected as the optimum reaction time. The concentrations of the substrate, such as catechol and H2O2, were crucial factors influencing the colorimetric signal and transmitted light intensity, and thus, the optimal concentrations of catechol and H2O2 were chosen to be 0.5% and 1.0 M, respectively (Fig. S3).



Fig. 4. Assay optimization with respect to transmitted light intensity with or without substrate. (A) Effect of the pH of working solution on detection of total enzyme. (B) Effect of the volume of MAb-PtPd NPs conjugations for the detection of the total enzyme. (C) Effect of reaction time after adding substrate for the detection of total enzyme. The concentration of BChE was 0.8 nM.

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for assay BChE activity was shown in Fig. S4. As shown in the Figure, the transmitted light intensity dramatically decreased at the first 5 min, and then became steady. So, 5 min was the optimum reaction time after adding the substrate. Analytical performance. As seen in Fig. 5A, the black color of the PtPd NPs on the test lines turned darker with the increase in the total amount of BChE under the optimal conditions because a sandwich-type model was adopted. However, with a low concentration of analyte, the test line was almost invisible. After the catechol coloration, the colorimetric signal was remarkably amplified, as seen in Fig, 5B, and the test line with a low concentration of BChE became clearly visible (brown). As ICTS was assayed on the ALS-based device, the transmitted light intensity decreased with the increase in the concentration of the total amount of BChE due to the absorbance of the brown polymerized product on the test line (Fig. 5D). The regression equation can be expressed as y = -452.9 ln(x) + 5632 (R2 = 0.9824), and the linear range was 0.05 nM6.4 nM with a detection limit of 0.025 nM. The stability of the ALS-based device was investigated by detecting the relative standard deviation values of the transmitted light intensity from the total amount of BChE at different concentrations (0.2 nM, 0.8 nM and 3.2 nM; Fig. S5A). All the relative standard deviation values of the transmitted light intensity were lower than 4%, which indicated the good reproducibility of the ALSdevice. Additionally, another stability assay for detecting the transmitted light intensity after 90 days was also conducted. Compared with the transmitted light intensity on day 1, the increase on day 90 was lower than 8%, which revealed that the good stability of the device (Fig. S5B). Similarly, an Ellman assay was conducted on the ALSbased ICTS to detect the BChE activity. With the increase in the BChE activity, the yellow color on the test line increased as the transmitted light intensity decreased (Fig. 5C). As seen in Fig. 5E, the regression equation could be expressed as y = 455.3 ln(x) +5950.2 (R2 = 0.9937), and the linear range was 0.1-6.4 nM with a detection limit of 0.028 nM. Assay of mock OP-BChE samples in human plasma. To demonstrate the application of the ALS-based device, mock samples containing active BChE and phosphorylated BChE (OP-BChE) were analyzed. Phosphorylated BChE was obtained by inhibiting active BChE with different concentrations of ethyl paraoxon in human plasma. As seen in Table 1, OPBChE samples were tested by detecting the total amount of BChE and the active BChE (three measurements per sample). The results showed that higher concentrations of ethyl paraoxon (0-64 nM) led to a higher phosphorylation degree (0.74-89.63%), which indicated the application potential of the proposed device. The recoveries of the total amount BChE measured by the ALS were from 94% to 106%, which indicated the high accuracy of this method. The relative deviation of these two methods was less than 7.01%, which also indicated an acceptable accuracy.

In the assay for the active BChE, the concentration of substrates was selected according to previous reports. 42,43 The pH value of the working solution was the same with the assay of total amount of BChE. The reaction time after adding substrate


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Fig. 5. Detection of total amount of BChE by using PtPd NPs as signal, before (A) and after (B) adding substrate catechol and H2O2. Detection of active enzyme after adding BTCh and DTNB (C). Concentration of BChE from 1-9 were 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 nM. The calibration curve of total amount of BChE(D) and active enzyme of BChE (E) recorded by ALS. The volume of the sample solution for each test is 70 µL. Table 1. Comparison phosphorylation of BChE in human plasma samples with two methods

Sample No.

1

2

3

4

5

6

7

8

Exposure to paraoxon-ethyl (nM)

0

1

2

4

8

16

32

64

Known BChE(nM)

4

4

4

4

4

4

4

4

Active BChE after exposure (c1,nM)

4.00

3.81

3.77

3.66

3.17

1.93

1.07

0.39

Total BChE by ALS (c2,nM)

4.03

3.95

4.01

4.05

4.00

4.24

3.90

3.76

phosphorylation of BChE by ALS(P%)1

0.74%

3.54%

5.99%

9.63%

20.75%

54.48%

72.56%

89.63%

phosphorylation of BChE by Ellman assay(P' %)

0%

3.42%

5.71%

9.20%

19.43%

50.91%

76.01%

92.34%

Relative deviations2

~

3.51%

+6.79%

+7.01%

-4.54%

-2.93%

+4.90%

+4.67%

1

(P%=(c2-c1)/c2×100%) 2 (Relative deviations = (P%-P’%)/P’%×100%)



CONCLUSION Based on the tetramer structure of BChE, only one MAb was utilized as both the recognition reagent and tracer probe. PtPd NPs were easily synthesized as nanozymes to develop a sandwich ICTS for evaluating the total amount of BChE. The brown color originating from the PtPd NPs-catechol coloration attenuated the light intensity dramatically, which could be detected by a low-cost, facile, homemade, 3D-printed ALSbased smartphone device. Additionally, the device could also be utilized to quantify the BChE activity based on Ellman assays. By subtracting the BChE activity from the total amount of BChE, the concentration of OP-BChE could be easily obtained for biomonitoring exposure to OPs. Due to the remarkable portability, low cost, short assay time and high sensitivity, the novel ALS-based ICTS shows great potential in various areas such as clinical diagnosis, environmental monitoring and food safety.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.” Absorption spectra of the catechol coloration and DTNB coloration. ELISA assay of BChE MAb for recognition of BChE and OP-BChE. Assay optimization with respect to concentration of catechol and H2O2. Effect of reaction time for detection of BChE activity. Stability of the ALS-based device. comparison the light intensity of the violet LED and blue LED as the illuminant.

AUTHOR INFORMATION Corresponding Author Email: [email protected] (D.D) Email: [email protected] (Y.L.)

Author Contributions 1

These authors contributed equally.

ACKNOWLEDGMENT This work was supported by the Centers for Disease Control and Prevention/National Institute for Occupational Safety and Health (CDC/NIOSH) Grant No. R21OH010768. Its contents are solely the responsibility of the authors and do not necessary represent the official views of CDC. YZ would like to acknowledge the China Scholarship Council for providing scholarship for working at WSU.

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