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Recombinant Peptidomimetic-Nano Luciferase Tracers for Sensitive Single-Step Immunodetection of Small Molecules Yuan Ding, Xiude Hua, He Chen, Fengquan Liu, Gualberto G. Gonzalez-Sapienza, and Ming-Hua Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04601 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Analytical Chemistry

Recombinant

Peptidomimetic-Nano

Luciferase

Tracers

for

Sensitive

Single-Step

Immunodetection of Small Molecules Yuan Ding †, ‡, Xiude Hua* , †, ‡, He Chen†, ‡, Fengquan Liu †, ‡, §, Gualberto González-Sapien⊥, Minghua Wang †, ‡ †

College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

‡

State & Local Joint Engineering Research Center of Green Pesticide Invention and Application,

Nanjing 210095, China §

Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing 210014, China

⊥

Cátedra de Inmunología, Facultad de Química, Instituto de Higiene, Universidad de la República,

Montevideo 11600, Uruguay * Tel.: +86 25 84395479. Fax: +86 25 84395479. E-mail address: [email protected] (X.H.).

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ABSTRACT Phage borne peptides isolated from phage libraries have proven to be valuable reagents for the development of small-molecule immunoassays. However, the large size, low diffusion rate and biological nature of the phage particles create some limitations to their use, and require secondary reagents for its detection. In this work, we explore the use of the Nano luciferase (NanoLuc) as fusion partner to generate recombinant tracers for immunoassay development. The imidaclothiz peptidomimetic C2-15 that specifically binds to the anti-imidaclothiz monoclonal antibody (mAb) 1E7, was fused to NanoLuc, both at the N terminus (C2-15-NanoLuc) and C terminus (NanoLuc-C2-15). NanoLuc-C2-15 showed better performance than C2-15-NanoLuc, and was adopted to develop a bioluminescent enzyme immunoassay (BLEIA) and a bioluminescence lateral flow immunoassay (BLLFIA) for imidaclothiz. The luminescence signal of NanoLuc-C2-15 rapidly reaches high intensity with slow attenuation, which enabled to capture the BLLFIA readout by using a smartphone without an external light source. The IC50 of the BLEIA and BLLFIA were 3.3 ± 0.2 and 6.4 ± 0.4 ng mL-1, respectively. Both immunoassays exhibited good accuracy for the detection of imidaclothiz in environmental and agricultural samples. Keywords: Nano luciferase; Phage borne peptide; Imidaclothiz; Bioluminescent enzyme immunoassay; Bioluminescence lateral flow immunoassay

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INTRODUCTION Phage display peptide libraries are useful tools for rapid selection of specific ligands for the development of immunoassay, pathogenic bacteria detection, cells targeted imaging and so on.1-6 For small molecule immunoassays, phage borne peptides are attractive alternative reagents for the development of a) competitive tests, in which an analyte peptidomimetic, also referred as mimotope, substitutes for the chemical hapten and/or the analyte,6, 7-9 or b) noncompetitive tests, where the phage borne peptide reacts specifically with the analyte-antibody immunocomplex but not with the uncomplexed antibody.1, 2, 10 In these applications, the strong signal associated to the detection of the large phage surface and the possibility of noncompetitive detection, typically result in assays with improved sensitivity, in the latter, the two-site recognition also provides better specificity.11, 12 However, there are also some limitations to the application of phage borne peptides. Owing to their large size (880 × 6-7 nm) and poor mobility, the phage particles displaying the peptide may not always be suitable for the development of rapid test and lateral flow immunoassay (LFIA). On the other hand, phage particles are uncommon in the immunoassay industry and their formulation as commercial reagents may be complicated by batch-to-batch variations in peptide expression, long-term stability issues and safety concerns related to their biological nature. To avoid these hassles, synthetic peptides chemically bound to carrier proteins, nanoparticles or to streptavidin through a biotin moiety,13-17 as well as recombinant peptide-protein chimeras,18-20 have been introduced to develop phage-free immunoassays. The use of recombinant chimeras is particularly attractive, because of the low cost that can be attained if their production is accomplished by bacterial fermentation. Moreover, if the protein partner is an active enzyme the peptide chimera could work directly as a tracer for one-step immunoassays.

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Nano luciferase (NanoLuc), an enzyme engineered for optimal ATP-independent bioluminescence emission, is an excellent partner to develop such tracers.21 NanoLuc derives from the Oluc-19 subunit of the deep-sea shrimp Oplophorus gracilirostris. In addition to its small size and monomeric nature, the enzyme was optimized for luminescence (~150 fold brighter than Firefly and Renilla luciferases), improved physical and chemical characteristics, and adaptation to the substrate furimazine, which is more stable than coelenterazine and less prone to autoluminescence.21 In this paper, we fused the cyclic 8-amino-acid peptidomimetic CLPPRMIYEC (C2-15) that specifically reacts with the anti-imidaclothiz monoclonal antibody (mAb) 1E7 to NanoLuc at the N terminus (C2-15-NanoLuc) and C terminus (NanoLuc-C2-15). Based on a competitive format, the recombinant chimeras were used to develop a bioluminescent enzyme immunoassay (BLEIA), and a bioluminescence lateral flow immunoassay (BLLFIA) that could be quantitated with the use of a smartphone. The accuracy of both tests was validated with real samples and by parallel analysis of samples by high-performance liquid chromatography (HPLC). MATERIALS AND METHODS Reagents. The pET-22b(+) vector, carbenicillin and Escherichia coli (E. coli) BL21(DE3) strain were purchased from Novagen (Darmstadt, GER). The pNL1.1 vector, Nano-Glo Luciferase Assay Substrate and EDTA (≥ 99.0%) were purchased from Promega (Madison, WI). Anti-His tag antibody was purchased from Abcam (Britain). The BCA Protein Assay Kit was purchased from Pierce (Washington, D.C.). The HisTrap HP column was purchased from GE Healthcare (Piscataway, NJ). Nitrocellulose membrane was purchased from Merck Millipore (Darmstadt, GER). The plano-convex lens was purchased from Thorlabs (Newton, NJ). The phage borne 4


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peptide C2-15 and mAb 1E7 were prepared previously.22, 23 Construction of the Vector. The vector pNL1.1 was employed as template to generate the C2-15-NanoLuc (the peptide at N terminus) and NanoLuc-C2-15 (the peptide at C terminus) genes by PCR using the primers F1, R1 and F2, R2, respectively (Figure 1). After digestion with EcoR I and Xho I and gel purification, the recombinant gene fragments were cloned into the pET-22b(+) vector containing the signal peptide pelB and a 6×His tag. The ligated vector was transformed into competent E. coli JM109 cells. Ten positive clones were randomly selected and sequenced with the T7 terminator primer. The plasmids containing the correct sequence were transformed into competent E. coli BL21(DE3) cells. The amino acid and DNA sequences of recombinant proteins are depicted in Figure S1, Supporting Information. Expression and Purification of the Recombinant Tracers. The E. coli BL21(DE3) cells were cultivated at 37 °C, 250 rpm in LB medium containing 50 µg mL-1 carbenicillin until the value of OD600 reached 0.6 AU. Then, the cultures were induced by addition of 0.1 mM IPTG (final concentration) and cultivated at 20 °C, 250 rpm overnight. The recombinant proteins were extracted from the periplasmic space by “osmotic shock”. Briefly, 40 mL cultures were collected and centrifuged at 10000 × g for 10 min. Then, 30 mL of 30 mM Tris-HCl (pH=8.0) containing 20% sucrose and 1 mM EDTA were applied to resuspend the cell pellet and were stirred at room temperature for 10 min. After centrifugation at 10000 × g for 10 min, the supernatant was removed, and the periplasmic proteins were released by addition of 30 mL of cold 5 mM MgSO4 and stirring for 10 min on ice. The periplasmic extracts were clarified by centrifugation (10000 × g) and purified by using an ÄKTA avant 25 on a 5 mL HisTrap HP column. The column was equilibrated with 10 column volumes of 0.5 M phosphate-buﬀered saline (PBS, pH=7.4) containing 5 mM 5



imidazol. Then, the extracts containing 5 mM imidazol were applied to the column. After washing with 10 column volumes of 0.5 M PBS containing 10 mM imidazol and 5 column volumes of 0.5 M PBS containing 50 mM imidazol. The target proteins were eluted with 5 column volumes of 0.5 M PBS containing 250 mM imidazol. The imidazol was removed by ultrafiltration. After measuring the concentration by BCA Protein Assay Kit, the protein solutions were supplemented with complete protease inhibitor and stored at -80 °C. BLEIA. White opaque microtiter plates (Corning Costar Corporation) were coated with mAb 1E7 (20 µg mL-1, 100 µL per well) in 0.14 M PBS by incubation overnight at 4 °C. After washing five times with PBS containing 0.05% Tween 20 (PBST), the plates were blocked with 5% skimmed milk (300 µL per well) in PBS for 2 h at 37 °C. After another washing step, recombinant protein in PBST containing 5% skimmed milk (2 µg, 50 µL per well) and imidaclothiz standard or prepared samples (containing 5 % methanol, 50 µL per well) were added to the plates and incubated at 37 °C for 1 h. After washing 10 times, 75 µL PBS and 25 µL Nano-Glo Luciferase assay buffer containing 0.5 µL Nano-Glo assay substrate were added to the wells and the values of bioluminescence at 450 nm were measured with a Spectra-Max M5 reader (Molecular Devices) with an integration time of 500 ms. BLLFIA. The BLLFIAs were performed on the nitrocellulose membrane (Hi-flow plus 135). The mAb 1E7 (3.6 mg mL-1) and anti-His tag antibody (1.5 mg mL-1) in PBS were dispensed as a test (T-line) and control (C-line), respectively. The nitrocellulose membranes were dried at 37 °C for 1 h, and then assembled with a sample pad (glass fiber pad) and absorbent pad. Assembled membranes were cut into 5 mm width strips and stored at room temperature until used. To quantitate the signal of the BLLFIA, the smartphone 3D printed accessory of bioluminescence 6


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signal detection for iPhone 6s were designed. The smartphone adaptor was contacted with LFIA cartridge by a plano-convex lens of 6 mm diameter (F=12 mm) in the lens holder (Figure 2). Fifty microliter imidaclothiz standard or samples (containing 5% methanol) were mixed with 50 µL of NanoLuc-C2-15 (containing 5 µg protein, 0.2% Tween 20 and 2 mg mL-1 BSA), and the mixture was dispensed into the LFIA sample inlet and let flow across the membrane for 15 min. Then, 100 µL of PBS were dispensed to wash the membrane for 10 min. The bioluminescence signal was obtained by adding 25 µL Nano-Glo Luciferase assay buffer containing 2 µL Nano-Glo Luciferase assay substrate to the nitrocellulose membrane from the optical window. The cartridge was inserted into the cartridge adaptor and the image was recorded using the smartphone camera after 20 s. The image was analyzed using the imageJ 1.46r software to quantitate the mean optical density of the T-line and adjacent T-line areas (background signal). The value of T-line was corrected by deducting the background signal. Cross-Reactivity (CR). The inhibitory concentration of the imidaclothiz related compounds causing 50% inhibition of the maximal signal (IC50) was used to calculate the CR according to the following formula: CR = [IC50 (imidaclothiz) / IC50 (analogue)] × 100%. Analysis of Spiked Samples. The blank samples of paddy water, soil, unpolished rice and orange were obtained from farms in Nanjing (Jiangsu province, China) and Taizhou (Zhejiang province, China). The samples were homogenized and spiked with known concentrations of imidaclothiz in methanol (the spiked final concentrations were 1, 10, 50 ng mL-1 for paddy water and 20, 100, 500 ng g-1 for other samples), and placed in the dark overnight at room temperature. The paddy water was detected directly by the BLEIA and BLLFIA after mixing with 50 µL of 2× buffer containing 7



2 µg and 5 µg NanoLuc-C2-15. Imidaclothiz in the other samples (10 g) was extracted with 20 mL of 25% methanol-PBS with vortexing for 5 min and sonication for 15 min. The supernatants were obtained by centrifugation at 1700 × g for 5 min and adjusted to 25 mL with the PBS in a volumetric flask, the concentrations of imidaclothiz were analyzed by the BLEIA and BLLFIA after appropriate dilution. HPLC Analysis and Validation. The unpolished rice real samples were extracted and analyzed by BLEIA, BLLFIA and HPLC. The samples for BLEIA and BLLFIA were prepared as described above. For HPLC analysis, 10 g of homogenized unpolished rice sample were extracted by 10 mL water and 40 mL acetonitrile with shaking for 1 h. After vacuum filtering, organic phase was separated by addition of 5 g NaCl, and 20 mL of the organic phase were evaporated to dryness by rotary evaporation. The concentrated extracts were dissolved in 2 mL of HPLC mobile phase and analyzed by HPLC (Agilent 1260) with an Eclipse pluse-C18 column (250 mm × 4.6 mm, 5µm). The mobile phase was methanol:water = 40:60 (v/v), flow rate was 0.7 mL min-1 at 30 °C. The detection wavelength was 270 nm and the injection volume was 20 µL. Statistical Evaluation. The standard curves of the BLEIA and BLLFIA for imidaclothiz were obtained by plotting the mean values of RLU or corrected intensity (y) versus the concentration of imidaclothiz (x) using the logistic equation of OriginPro 8.0. Correlation curves between the immunoassays and HPLC were obtained by plotting the detection results of HPLC (y) versus those of BLEIA or BLLFIA (x) using OriginPro 8.0. Significant difference between the detection results of HPLC and those of immunoassays was assessed by using Student’s t test. Statistical significance was set at least 0.05 (P＞0.05). Statistical analyses were performed using the DPS 7.05. 8


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RESULTS AND DISCUSSION Characterization of Recombinant Tracers. Figure 1A shows the schematic representation of the recombinant bioluminescent tracers. Owing to the presence of a disulfide bond in C2-15, the periplasmic compartment of E.coli was employed to produce the recombinant proteins. The NanoLuc tracers were present in the periplasmic extract as soluble proteins. The SDS-PAGE analysis of the purified tracers showed that the molecular masses were about 23 kDa (Figure 3A). Besides, the tracers were detected with an anti-His tag antibody in western blots (Figure 3B). The luminescence spectrum of the recombinant proteins had a maximum emission wavelength of 450 nm (Figure 3C), which was not affected by the relative position of the peptide and is in agreement with the reported spectral maximum (454 nm) of NanoLuc.21 The functional binding of the recombinant tracers was assessed by surface plasmon resonance using a Biacore T200 instrument. The dissociation constants (KD) of the interaction of the tracers with the mAb 1E7 were 1.04×10-6 M and 6.60×10-7 M for the peptides fused at the N- and C-terminus of the recombinant proteins, respectively (Figure S2). The lower KD of C2-15-NanoLuc was somehow unexpected considering that this peptide was originally selected as an N-terminal fusion of the phage protein pIII. Nevertheless, in spite of this difference, both constructs gave place to similar assay sensitivity when used as tracers in bioluminescent ELISA, IC50 = 7.7 ± 0.2 and 5.7 ± 0.2 ng mL-1 for C2-15-NanoLuc and NanoLuc-C2-15, respectively (Figure S3). Both tracers performed with similar sensitivity, but due to the higher affinity of the latter, lower concentrations are needed to achieve the same luminescence signal value and for this reason, NanoLuc-C2-15 was chosen to develop the bioluminescent assays. Bioluminescence Kinetics. Figure 4A shows a typical readout acquired by using the smartphone 9



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detection system, and the intensity curves generated by the image analysis software. The bioluminescence intensity profiles measured for the T-line and C-line did not interfere with each other, showing that they can be quantitatively measured by using the smartphone reader. Typically, luminescence associated to luciferases requires a short time to reach a maximal emission, which is followed by progressive signal decay. To assess the luminescence signal of NanoLuc-C2-15 in immunoassays, the signal kinetics of BLEIA and BLLFIA were studied. Initially, BLEIA and BLLFIA were run with PBS in the absence of imidaclothiz, and the luminescence signals were continuously measured with a Spectra-Max M5 reader (Molecular Devices) and smartphone, respectively. In the case of BLEIA, the signal reached the maximum (RLUmax=223302) immediately and only decreased by 18 % after 30 min (Figure 4B), while in the case of BLLFIA, the signal reached a maximum (corrected intensitymax=31.0) after 20 s, stabilized for about 1 min, and decreased steadily with a signal half-life of about 3 min (Figure 4C and 4D). The signal of BLLFIA was read from the nitrocellulose membrane after adding the Nano-Glo Luciferase assay substrate, which rapidly spread on the nitrocellulose membrane and soon started to dry. On the contrary, the signal of BLEIA was obtained from the microtiter plate, which provided a stable reaction condition and therefore a longer signal half-life. Compared with other bioluminescent proteins or chemiluminescent substrates,22,

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the luminescence signal of

NanoLuc-C2-15 rapidly reaches high intensity with slow attenuation. These results indicated that NanoLuc-C2-15 had great promise for the development of immunoassays with high sensitivity and short detection time. Optimization of Methanol Concentration. Methanol, which was used to dissolve the analyte, can interfere with the antibody-antigen reaction, so the influence of this solvent was studied. The 10


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test was sensitive to this solvent and the maximum tolerated concentration (final concentration) of methanol for BLEIA and BLLFIA was 2.5 % (Figure S4). Standard Curves of BLEIA and BLLFIA. The imidaclothiz standard curves of the BLEIA and BLLFIA are shown in Figure 5A. The IC50 value, limit of detection (LOD) and linear range (IC10 to IC90) of the BLEIA were 3.3 ± 0.2, 0.2 and 0.2 to 57.3 ng mL-1, while the BLLFIA were 6.4 ± 0.4, 0.3 and 0.3 to 81.7 ng mL-1, respectively. The reproducibility was evaluated by the relative standard deviations (RSDs) of the standard curve data that were measured by three independent operators. The RSDs of BLEIA and BLLFIA were in the ranges of 4.0% to 9.3% and 5.2% to 11.5%, respectively (Table S1). Figure 5B shows the images of BLLFIA for serial concentrations of imidaclothiz. When the concentration of imidaclothiz increased to 6 ng mL-1, the brightness of T-line was significantly weaker than that at zero concentration, being the LOD of BLLFIA for the pesticide. Considering that the maximum residue limits (MRL) of imidaclothiz permitted in China are 0.3, 0.2, 0.1 and 0.2 mg kg-1 for tea, orange, brown rice and wheat (GB 2763-2014), respectively, the sensitivities of the BLEIA and BLLFIA meet the requirements for the determination of this pesticide. The sensitivity of both methods was roughly the same, and very similar to the sensitivity of the phage ELISA (4.02 ng mL-1).22 This shows that the transference of the peptide from the phage to the recombinant protein did not affect its activity, showing that the NanoLuc-peptide tracer works as an efficient substitute of the phage particles, avoiding the shortcomings associated to their large size, low diffusion rate and biological nature. Based on the same mAb 1E7, the sensitivities of the BLEIA and BLLFIA were improved 26-fold and 13-fold with regard to the conventional ELISA (IC50=87.5 ng mL-1) developed by the chemical synthetic antigen.23 In addition, the recombinant 11



protein could be used as a tracer to avoid the use of secondary reagents for detection, which made the BLEIA and BLLFIA faster and simpler than conventional ELISA and phage ELISA. It is also interesting to note that the sensitivity of phage ELISA is always improved by the large amount of tracer (typically an anti-phage antibody coupled to HRP) that is associated to the extended surface of the phage.7 This cumulative signal is lost when the peptide is transferred to the chimeric protein, however the assay sensitivity was not affected. It seems that this loss of signal is compensated in the chimeric tracer by the high luminescence efficiency of the NanoLuc. Specificity of BLEIA and BLLFIA. To test the specificity of the assays, the cross-reactivity (CR) of a panel of imidaclothiz related neonicotinoids was tested in the luminescent assays. Except for imidacloprid that shares the nitro-dihydroimidazol-amine group (CR = 93.6% for BLEIA and 95.1% for BLLFIA), there were no significant CR (≤2.30%) with the other analogues (Table 1). As expected, this cross-reactivity pattern was the same found for the phage-ELISA set up with the phage borne C2-15 (data not shown). Analysis of Spiked Samples. Preliminary experiments were performed to estimate the interference of different matrices in the assays and the sample dilution necessary to eliminate it. Paddy water samples could be tested after 2-fold dilution by both assays, but soil, unpolished rice and orange samples required respectively 40-, 20- and 40-fold dilutions for BLEIA, and 20-, 40and 20- fold dilutions for BLLFIA (Figure S5 and S6). The spiked concentrations were set to 1, 10, 50 ng mL-1 for paddy water and 20, 100, 500 ng g-1 for the other samples. The mean recoveries and RSDs for BLEIA were 79.0-104.1% and 2.3-10.9%, and 78.0-110.2% and 2.3-10.3% for BLLFIA, respectively (Table 2). The images acquired with the smartphone of BLLFIAs for the analysis of spiked samples showed the brightness of T-line was reduced with the increase of 12


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spiked concentration (Figure S7). HPLC Validation. The analytical performances of the two bioluminescence methods were also compared to HPLC for the determination of imidaclothiz. Initially, the HPLC method was optimized using an imidaclothiz-free unpolished rice sample (Figure S8). Average recoveries were in the range of 82.7% to 94.7% with RSDs of 2.1% to 3.2% (Table S2). The BLEIA, BLLFIA and HPLC methods were then used to analyze unpolished rice real samples, the amounts of imidaclothiz detected by the immunoassays were no significantly different to those detected by HPLC because the P values were greater than 0.05 (0.7098 for BLEIA, 0.5607 for BLLFIA) (Table S3). In addition, there was an excellent correlation of both immunoassays with the instrumental method, which was evident by the values of the slop that were very close to 1 (Figure 6). These results indicated that the presented immunoassays were reliable and accurate for the determination of imidaclothiz in real samples. CONCLUSIONS In this study, we demonstrated that peptidomimetics of small analytes isolated from phage display libraries can be used as chimeric constructs with the NanoLuc to produce recombinant reagents to develop phage-free immunoassays. This is a significant contribution to the field of small-molecule immunoassays because a) there is no need of chemical conjugation of the actual analyte, b) the stoichiometry (peptide/enzyme) is fixed with no batch-to-batch variations, and c) the final tracer enables the development of single-step immunoassays with fast readouts. To the best our knowledge, there is only one previous report of such construct, but in that report the authors used E. coli alkaline phosphatase as fusion partner, which in spite of being mutated to increase its kcat required hours of incubation with the substrate to generate a measurable signal.27 13



Owing to the high luminous efficiency of NanoLuc, the peptide-luciferase chimera can be efficiently used as tracer providing high assay sensitivity. The direct linkage of the enzymatic activity to the peptidomimetic avoids the use of additional secondary reagents for detection, making it possible to develop shorter one-step immunoassays. The tracer was efficiently produced in soluble form by shake cultivation, with typical yields of 2.3 mg per liter, which corresponds to the analysis of about 1200 samples by BLEIA or 460 samples in the case of BLLFIA, enabling the production of low-cost immunoassays. The brightness of NanoLuc also allowed the development of a lateral flow test that could be read by using a smartphone equipped with a 3D-printed detection accessory, without the need of an external light source. The immunoassays developed with the bioluminescent tracer performed with very good recoveries when tested with different matrices and showed excellent correlation with the instrumental method. Since the use of phage display peptide libraries for the selection of peptidomimetics and anti-inmunocomplex peptides is increasingly used, this novel one-step recombinant tracer will be a wonderful addition to develop phage-free peptide-based rapid test. AUTHOR INFORMATION Corresponding Author * Tel.: +86 25 84395479. Fax: +86 25 84395479. E-mail address: [email protected] (X.H.). Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31431794), the National Key Research and Development Program of China (2017YFF0210201), the National 14


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Natural Science Foundation of China (31772194), and CSIC 149 UdelaR Uruguay. ASSOCIATED CONTENT Supporting Information The detailed result of amino acid and DNA sequences of recombinant proteins; the comparison between C2-15-NanoLuc and NanoLuc-C2-15 including affinity and sensitivity; the effect of methanol on BLEIA and BLLFIA; the reproducibility of the BLEIA and BLLFIA; the effect of matrix interference on BLEIA and BLLFIA; the bioluminescence images of spiked samples at different imidaclothiz concentrations; the representative chromatograms of HPLC; the recoveries of samples spiked with imidaclothiz by HPLC; the comparison of imidaclothiz residues between the BLEIAs, BLLFIAs and HPLC in real samples. REFERENCES (1) González-Techera, A.; Kim, H. J.; Gee, S. J.; Last, J. A.; Hammock, B. D.; González-Sapienza, G. Anal. Chem. 2007, 79, 9191−9196. (2) Arola, H. O.; Tullila, A.; Kiljunen, H.; Campbell, K.; Siitari, H.; Nevanen, T. K. Anal. Chem. 2016, 88, 2446−2452. (3) Ono, K.; Takeuchi, K.; Ueda, H.; Morita, Y.; Tanimura, R.; Shimada, I.; Takahashi, H. Angew. Chem. Int. Ed. 2014, 53, 2597−2601. (4) Ma, K.; Wang, D. D.; Lin, Y.; Wang, J.; Petrenko, V.; Mao, C. Adv. Funct. Mater. 2013, 23, 1172−1181. (5) Günay, K. A.; Benczédi, D.; Herrmann, A.; Klok, H. A. Adv. Funct. Mater. DOI: 10.1002/adfm.201603843. (6) Arévaloa, F. J.; González-Techera, A.; Zona, M. A.; González-Sapienza, G.; Fernández, H. Biosens. Bioelectron. 2012, 32, 231−237. (7) Cardozo, S.; González-Techera, A.; Last, J. A.; Hammock, B. D.; Kramer, K.; González-Sapienza, G. Environ. Sci. Technol. 2005, 39, 4234−4241. (8) González-Techera, A.; Umpiérrez-Failache, M.; Cardozo, S.; Obal, G.; Pritsch, O.; Last, J. A.; 15



Gee, S. J.; Hammock, B. D.; González-Sapienza, G. Bioconjug. Chem. 2008, 19, 993−1000. (9) Kim, H. J.; González-Techera, A.; González-Sapienza, G.; Ahn, K. C.; Gee, S. J.; Hammock, B. D. Environ. Sci. Technol. 2008, 43, 2047−2053. (10) Lim, S. L.; Ichinose, H.; Shinoda, T.; Ueda, H. Anal. Chem. 2007, 79, 6193−6200. (11) Hua, X. D.; Zhou, L. L.; Feng, L.; Ding, Y.; Shi, H. Y.; Wang, L. M.; Gee, S. J.; Hammock, B. D.; Wang, M. H. Anal. Chim. Acta 2015, 890, 150−156. (12) Rossotti, M. A.; Carlomagno, M.; González-Techera, A.; Hammock, B. D.; Last, J.; González-Sapienza, G. Anal. Chem. 2010, 82, 8838−8843. (13) Hwang, H. J.; Ryu, M. Y.; Park, C. Y.; Ahn, J.; Park, H. J.; Choi, C.; Ha, S. D.; Park, T. J.; Park, J. P. Biosens. Bioelectron. 2017, 87, 164−170. (14) Liu, X.; Xu, Y.; He, Q. H.; He, Z. Y.; Xiong, Z. P. J. Agric. Food Chem. 2013, 61, 4765−4770. (15) Peltomaa, R.; Benito-Peña, E.; Barderas, R.; Sauer, U.; González Andrade, M.; Moreno-Bondi, M. C. Anal. Chem. 2017, 89, 6217−6224. (16) Vanrell, L.; Gonzalez-Techera, A.; Hammock, B. D.; Gonzalez-Sapienza, G. Anal. Chem. 2013, 85, 1177−1182. (17) Yeh, C. Y.; Hsiao, J. K.; Wang, Y. P.; Lan, C. H.; Wu, H. C. Biomaterials 2016, 99, 1−15. (18) Carlomagno, M.; Lassabe, G.; Rossotti, M.; González-Techera, A.; Vanrell, L.; González-Sapienza, G. Anal. Chem. 2014, 86, 10467−10473. (19) Lassabe, G.; Rossotti, M.; González-Techera, A.; González-Sapienza, G. Anal. Chem. 2014, 86, 5541−5546. (20) Xu, Y.; He, Z.; He, Q.; Qiu, Y.; Chen, B.; Chen, J.; Liu, X. J. Agric. Food Chem. 2014, 62, 8830−8836. (21) Hall, M. P.; Unch, J.; Binkowski, B. F.; Valley, M. P.; Butler, B. L.; Wood, M. G.; Otto, P.; Zimmerman, K.; Vidugiris, G.; Machleidt, T.; Robers, M. B.; Benink, H. A.; Eggers, C. T.; Slater, M. R.; Meisenheimer, P. L.; Klaubert, D. H.; Fan, F.; Encell, L. P.; Wood, K. V. ACS Chem. Biol. 2012, 7, 1848−1857. (22) Ding, Y.; Hua, X. D.; Sun, N. N.; Yang, J. C.; Deng, J. Q.; Shi, H. Y.; Wang, M. H. Sci. Total Environ. 2017, 609, 854−860. (23) Fang, S.; Zhang, B.; Ren, K. W.; Cao, M. M.; Shi, H. Y.; Wang, M. H. J. Agric. Food Chem. 16


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2011, 59, 1594−1597. (24) Lim, C. K.; Lee, Y. D.; Na, J.; Oh, J. M.; Her, S.; Kim, K.; Choi, K.; Kim, S.; Kwon, I. C. Adv. Funct. Mater. 2010, 20, 2644−2648. (25) Yu, X. Z.; Wen, K.; Wang, Z. H.; Zhang, X. Y.; Li, C. L.; Zhang, S. X.; Shen, J. Z. Anal. Chem. 2016, 88, 3512−3520. (26) Roda, A.; Michelini, E.; Cevenini, L.; Calabria, D.; Calabretta, M. M.; Simon, P. Anal. Chem. 2014, 86, 7299−7304. (27) Gonzalez-Techera, A.; Umpierrez-Failache, M.; Cardozo, S.; Obal, G.; Pritsch, O.; Last, J. A.; Gee, S. J.; Hammock, B. D.; Gonzalez-Sapienza, G., Bioconjug. Chem. 2008, 19, 993−1000.

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FIGURE CAPTIONS Figure 1. Schematic diagrams of the recombinant tracers and primers. (A) The constructs of the recombinant C2-15-NanoLuc and NanoLuc-C2-15, C2-15 contains a disulfide bond to form a cyclic peptide. (B) Primers used to generate the genes for C2-15-NanoLuc and NanoLuc-C2-15. The regions coding for the peptide, spacers (Sp), and the annealing to the NanoLuc gene are denoted below the nucleotide sequence. Figure 2. The 3D printed detection accessory (resin) of BLLFIA. (A) The cartridge for the LFIA strip. (B) The smartphone accessory. (C) A smartphone with the detection accessory. Figure 3. Expression analysis of the recombinant tracer by coomassie-stained SDS-PAGE, western blot and emission spectrum. (A) Recombinant proteins purified on HisTrap HP columns, C2-15-NanoLuc (lane 1) and NanoLuc-C2-15 (lane 2). (B) Western blot analysis of C2-15-NanoLuc and NanoLuc-C2-15. (C) The luminescence spectra of C2-15-NanoLuc and NanoLuc-C2-15 at the same concentration. Figure 4. Luminescence signal of NanoLuc-C2-15. (A) Bioluminescence intensity profile of BLLFIA T- and C-line acquired with the smartphone reader and analyzed with ImageJ. (B) Kinetic curve of bioluminescence for BLEIA. (C) Kinetic curve of bioluminescence for BLLFIA. (D) Bioluminescence images acquired with the smartphone corresponding to the time points shown in (C). Figure 5. Performance of the NanoLuc tracers in BLEIA and BLLFIA formats. (A) BLEIA (●) and BLLFIA (■) imidaclothiz standard curves. (B) Bioluminescence images acquired with the smartphone corresponding to the standard curves of BLLFIA in (A). Figure 6. Correlations between the immunoassays and HPLC. Line equation and correlation

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coefficient of imidaclothiz determination in unpolished rice samples by BLEIA and HPLC (A) and BLLFIA and HPLC (B). Table 1. Cross-Reactivity of Analogues Structurally Related to Imidaclothiz Determined by BLEIA and BLLFIA Table 2. Average Recoveries of Imidaclothiz Spiked into Environmental and Agricultural Samples (n=3)

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Table 1. Cross-Reactivity of Analogues Structurally Related to Imidaclothiz Determined by BLEIA and BLLFIA BLEIA BLLFIA Compound Chemical structure -1 IC50 (ng mL ) CR (%) IC50 (ng mL-1) CR (%) Imidaclothiz

3.3

100

6.4

100

Imidacloprid

3.6

93.6

6.7

95.1

Thiacloprid

184.2

1.8

277.5

2.3

Clothianidin

569.2

0.6

1132.5

0.6

Acetamiprid

537.1

0.6

2798.9

0.2

Thiamethoxam

1242.7

0.3

3302.3

0.2

Nitenpyram

>10000

10000

10000

10000

Recombinant Peptidomimetic-Nano Luciferase ... - ACS Publications

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