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M13 Bacteriophage/Silver Nanowire Surface-Enhanced Raman Scattering Sensor for Sensitive and Selective Pesticide Detection Eun Hye Koh, ChaeWon Mun, Chun Tae Kim, Sung-Gyu Park, Eun Jung Choi, Sun Ho Kim, Jaejeung Dang, Jaebum Choo, Jin-Woo Oh, Dong-Ho Kim, and Ho Sang Jung ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01470 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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ACS Applied Materials & Interfaces

M13 Bacteriophage/Silver Nanowire Surface-Enhanced Raman Scattering Sensor for Sensitive and Selective Pesticide Detection Eun Hye Koh†,#, ChaeWon Mun†, ChunTae Kim#, Sung-Gyu Park†, Eun Jung Choi#, Sun Ho Kim†, Jaejeung Dang§, Jaebum Choo§, Jin-Woo Oh#, Dong-Ho Kim†,* and Ho Sang Jung†,*

†

Advanced Functional Thin Films Department, Korea Institute of Materials Science (KIMS),

Changwon, Gyeongnam, 51508, Republic of Korea #

Department of Nano Fusion Technology, Pusan National University (PNU), Busan, 46241, Republic of

Korea §

Department of Bionano Technology, Hanyang University, Ansan, 426-791, Republic of Korea

CORRESPONDING AUTHORS E-mail: [email protected] (Dr. Dong-Ho Kim) E-mail: [email protected] (Dr. Ho Sang Jung)

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ABSTRACT A surface-enhanced Raman scattering (SERS) sensor comprising silver nanowires (AgNWs) and genetically engineered M13 bacteriophages expressing a tryptophan-histidine-tryptophan (WHW) peptide sequence (BPWHW) was fabricated by simple mixing of BPWHW and AgNW solutions, followed by vacuum filtration onto a glass-fiber filter paper (GFFP) membrane. The AgNWs stacked on the GFFP formed a high density of SERS-active hot spots at the points of nanowire intersections, and the surface-coated BPWHW functioned as a bioreceptor for selective pesticide detection. The BPWHWfunctionalized AgNW (BPWHW/AgNW) sensor was characterized by scanning electron microscopy (SEM), confocal scanning fluorescence microscopy (CSFM), atomic force microscopy (AFM) and Fourier transform infrared (FT-IR) spectroscopy. The Raman signal enhancement and the selective pesticide SERS detection properties of the BPWHW/AgNW sensor were investigated in the presence of control substrates such as wild-type M13 bacteriophage-decorated AgNWs (BPWT/AgNW) and undecorated AgNWs (AgNW). The BPWHW/AgNW sensor exhibited a significantly higher capture capability for pesticides, especially paraquat (PQ), than the control SERS substrates, and it also showed a relatively higher selectivity for PQ than for other bipyridylium pesticides such as diquat (DQ) and difenzoquat (DIF). Furthermore, as a field application test, PQ was detected on the surface of PQpretreated apple peels, and the results demonstrated the feasibility of using a paper-based SERS substrate for on-site residual pesticide detection. The developed M13 bacteriophage-functionalized AgNW SERS sensor might be applicable for the detection of various pesticides and chemicals through modification of the M13 bacteriophage surface peptide sequence.

KEYWORDS M13 Bacteriophage, Silver Nanowires, Surface Enhanced Raman Scattering, Chemical Sensor, Pesticide Detection ACS Paragon Plus Environment

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1. INTRODUCTION Surface-enhanced Raman scattering (SERS) technologies have been widely applied for various molecular sensors because of their great Raman signal enhancement capability, which has enabled the development of ultrasensitive molecular detection sensors. The electromagnetic field enhancement between novel metal nanostructures, areas known as “hot spots”, is the main effect of Raman signal amplification.1 Furthermore, the enhanced Raman signal exhibits the intrinsic chemical spectrum of the detected molecule, enabling the development of label-free chemical sensors.2 However, for the selective capture and detection of target molecules using SERS sensors, receptor functionalization on the sensor surface is required, especially around the hot spots. Various receptors, such as antibodies, aptamers, and macrocycles, have been used for functionalization of the surface of SERS materials.3-6 In the case of antibodies, because of the abundant established library of antigen-antibody information, various antibody-decorated SERS biosensors have been developed for target-specific biomarker detection. However, due to the size limitation of the antibody, it is difficult to place an antibody at a hot spot smaller than the antibody. Additionally, Raman reporter dyes or additional plasmonic nanoparticles are usually required because the relatively small Raman cross section of the target proteins impedes labelfree sensor design.7 On the other hand, receptors such as aptamers and macrocycles can decorate hot spot areas, but the limited number of known aptamer sequences and macrocycle-target pairs are an obstacle for the development of widely applicable SERS biosensors. The M13 bacteriophage is a one-dimensional (1D) wire-shaped virus that expresses 2700 copies of pVIII surface proteins.8 The M13 bacteriophage can be produced in Escherichia coli and exhibits a regular shape with lengths and diameters of 880 and 6.6 nm, respectively.9-10 Because of the well-known surface protein sequences, the M13 bacteriophage can be genetically modified to exhibit different surface protein properties from those of the wild-type.11 Various biosensors, drug delivery systems, and scaffolds have been developed by exploiting the advantages of the M13 bacteriophage.10,

12-14

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demonstrated to enhance the sensitivity and selectivity of such sensors for the target molecule when used as bioreceptors.11 Although the known receptor sequences are limited and under development, M13 bacteriophages have great advantages as bioreceptors over antibodies and aptamers, such as better mass productivity, lower production costs, and a large density of binding sites.8,11,15 In this work, on the basis of our previous report of silver nanowire plasmonic nanomaterials,1,16 an M13 bacteriophage-functionalized SERS sensor was developed for selective pesticide detection. The genetically engineered M13 bacteriophage was decorated on the AgNW surface, and the network structure of AgNWs was formed on glass-fiber filter paper (GFFP) (Scheme 1). The random stacking of AgNWs created a high density of hot spots and a significantly enhanced Raman signal. Additionally, surface-functionalized M13 bacteriophages expressing tryptophan-histidine-tryptophan (WHW) peptide sequences (BPWHW) functioned as bioreceptors for the selective detection of paraquat (PQ). Field application tests using a hand-held Raman spectrometer showed the possibility of on-site residual pesticide detection system development. To the best of our knowledge, this work is the first demonstration of a SERS sensor comprising M13 bacteriophages and AgNWs.


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2. EXPERIMENTAL SECTION 2.1. Materials. AgNW solution at a concentration of 0.3 wt% was obtained from C3NANO Co., Ltd. (Cheongju, South Korea). Glass-microfiber filter paper with a diameter of 47 mm was purchased from GE Healthcare (Tokyo, Japan). Methyl viologen dichloride hydrate (PQ), difenzoquat methyl sulfate (DIF), diquat dibromide monohydrate (DQ), MPP+ iodide (cyperquat, CQ), rhodamine 6G (R6G), and Bradford assay reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-M13 bacteriophage coat protein g8p antibody and goat anti-mouse IgG H&L antibody (Alexa Fluor® 488) were purchased from Abcam (Cambridge, MA, USA). 2.2. Preparation of genetically modified bacteriophages. The major coat proteins of the M13 bacteriophage were modified with a WHWQ sequence (Trp (W)-His (H)-Trp (W)-Gln (Q)) using subcloning techniques described elsewhere.17 The pVIII forward primer was designed to include the Pst I

restriction

site,

and

the

insert

sequence

was

5

′

-

ATATATCTGCAGNKNNKTGGCATTGGCAGNNKNNKGATCCCGCAAAAGCG GCCTTTAACTCCC-3′. The reverse primer was designed as 5′-GCTGTCTTTCGCTGCAGAGGGTG-

3′ to make the vector linear and complimentary to the engineered gVIII 3′-5′ region. PCR verification was not performed by the authors in this manuscript. The insertion of DNA into phage clones (WHW) was carried out, and the clones were kindly donated from Prof. Seung-Wuk Lee’s laboratory at the Department of Bioengineering, University of California, Berkeley, California 94720, USA. 2.3. Preparation of M13 bacteriophage-functionalized AgNW SERS sensors. AgNW solution (DI water) with a concentration of 0.15 wt% was mixed with a solution of WHW-expressing or WT M13 bacteriophages (100 µg/mL) at a ratio of 1:1. After 5 min, 3 mL of the mixed solution was vacuum filtered through a glass-fiber filter membrane for 1 min. The filter papers containing the prepared AgNW and M13 bacteriophage mixture were dried in a vacuum desiccator at room temperature for 24 h. A ACS Paragon Plus Environment

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control AgNW SERS substrate was prepared by mixing DI water instead of the M13 bacteriophage solution. 2.4. Characterization of the M13 bacteriophage/AgNW SERS substrate. The surface morphologies of the AgNW substrate, BPWT/AgNW and BPWHW/AgNW were investigated using field-emission scanning electron microscopy (FE-SEM, JSM-6700F, JEOL). The nanoscale surface morphologies of AgNW and BPWHW/AgNW were observed by atomic force microscopy (AFM, NX10, Park System). The IR spectra of the AgNW, BPWHW, BPWHW/AgNW and BPWHW/AgNW formed after centrifugation (3000 rpm, 10 min) were obtained by Fourier transform infrared spectroscopy (FT-IR, ID5 ATR, Sinco Nicolet). The decorated BPWHW structure in the AgNW network was observed using a confocal scanning fluorescence microscopy system (TCS-SP5-MP-SMD confocal system, Leica Microsystems) after immunofluorescence staining using anti-M13 bacteriophage coat protein g8p primary antibodies, followed by anti-mouse IgG (Alexa Fluor® 488) secondary antibody labeling. 2.5. Raman signal enhancement characterization of BPWHW/AgNW. The Raman signal enhancement of the prepared AgNW, BPWT/AgNW, and BPWHW/AgNW substrates was tested after dropping 20 µL of an R6G solution at various concentrations (1000, 500, 100, 50, and 10 nM) on each substrate. The Raman signal was obtained using an optical Raman system (Nanoscope Systems, Inc., Daejeon, South Korea) with the following measurement conditions: an exposure time, a laser power, and a laser wavelength of 5 s, 6 mW and 633 nm, respectively. All of the spectra were collected five times at random points from the three sets of samples prepared under the same conditions (n = 3). The spectra were averaged for plotting. 2.6. Binding capacity test of BPWHW and BPWT for PQ. The Bradford assay was carried out to adjust the concentrations of BPWHW and BPWT and to check for the absence of M13 bacteriophage in the supernatant after centrifugation at 12000 rpm for 15 min. Then, 1 mL each of BPWHW and BPWT at a concentration of 0.1 mg/mL was mixed with PQ at a final concentration of 5 ppm and then left at ACS Paragon Plus Environment

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room temperature for 1 h. The UV-vis absorbance spectra of the PQ mixture with BPWHW and BPWT before and after centrifugation were measured. The change in PQ amount was calculated according to the standard curve at various PQ concentrations. The adhered PQ amount for each unit weight of BPWHW and BPWT was calculated by dividing the PQ weight change by the weight of each M13 bacteriophage in 1 mL of solution. 2.7. Sensitivity and selectivity tests of the BPWHW/AgNW SERS sensor. In the sensitivity test of BPWHW/AgNW for pesticides, the limit of detection was investigated after dropping 20 µL of a PQ solution at various concentrations (100, 50, 10, 5, and 1 ppm). The Raman signal was measured after complete drying of the solvent. In the selectivity test of BPWHW/AgNW, the Raman signal change before and after the pesticide (PQ, DQ and DIF) washing step was compared with that measured for the BPWT/AgNW and AgNW substrates. The washing procedure consisted of shaking the pesticidecontaining substrates in 10 mL of fresh DI water for 1 min on an orbital shaker at 100 rpm (CWS-350, Jeio Tech.). The capture efficiency for each pesticide was calculated by the following formula after baseline correction for the measured intensity: Efficiency (%) = (Ri - ∆R)/Ri, (∆R = Ri - Rf), where Ri is the Raman intensity before pesticide washing and Rf is the Raman intensity after pesticide washing. Further, Raman mapping of PQ on BPWHW/AgNW, BPWT/AgNW and AgNW before and after the washing step was carried out using a Renishaw 2000 Raman microscope system (Renishaw plc, UK) within a 105 µm × 105 µm area with a laser wavelength of 633 nm, an exposure time of 0.5 s, and a laser power of 15 mW. 2.8. Field application test using a hand-held Raman spectrometer. Twenty microliters of PQ solution samples at 50, 25, 10, 5, and 1 ppm were dropped onto the surface of freshly washed apples and allowed to dry for 24 h. The absorbed mass quantity of PQ was calculated as 1, 0.5, 0.2, 0.1 and 0.02 µg, respectively, after complete drying. The surface of each pesticide-treated apple sample was subsequently wiped on a 1 cm2 BPWHW/AgNW sensor wetted with 20 µl of DI water. The Raman signal was measured directly on the BPWHW/AgNW sensor after complete drying of the transferred pesticide. The ACS Paragon Plus Environment

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signal was measured using a hand-held Raman spectrometer (CBEx-M, Snowy Range Instruments®, USA) with the exposure time, laser power, and laser wavelength set at 3 s, 10 mW and 638 nm, respectively.

3. RESULTS AND DISCUSSION 3.1. Characterization of the BPWHW/AgNW substrate. The surface morphology of the BPWHW/AgNW structure was investigated by FE-SEM. As shown in Figure 1a, a multistacked AgNW network structure was observed with a random orientation on the GFFP. Because the pore size of the GFFP was 700 nm and the length of AgNWs was 18 ± 2 µm, BPWHW-functionalized AgNWs were successfully filtered on the GFFP, as shown in Figure 1b. The multistacked AgNWs formed a high density of hot spots during the vacuum filtration procedure. However, because the M13 bacteriophages exhibited a thin diameter (ca. 6.6 nm) and the material was low density, the structure was difficult to observe by SEM. The SEM images of AgNW, BPWT/AgNW (Figure S1a and S1b), and BPWHW/AgNW (Figure 1a) showed that the structure of the AgNW network was not altered by the introduction of M13 bacteriophages. The network structure of BPWHW/AgNW was also observed by confocal laser scanning microscopy after immunofluorescence staining of M13 bacteriophages using anti-bacteriophage g8p surface protein primary antibody and Alexa Fluor 488-labeled secondary antibody. Figure 1c shows the green fluorescence of M13 bacteriophages coated on the surface of AgNWs. The strong fluorescence signal was similar to the signal from the AgNW network structure because of the M13 bacteriophage adhesion along the AgNW surface during the mixing and vacuum filtration procedure. No significant fluorescence signal was observed from the control AgNW substrate (Figure 1d). The nanoscale surface morphology of the BPWHW/AgNW was investigated by AFM. As shown in Figure 2b, the adhered M13 bacteriophage layer on the AgNW surface was observed along the direction of the AgNWs, whereas the control AgNW surface showed clean lateral faces (Figure 2a). The surface of the AgNWs became rough and formed a thorn-like structure after BPWHW functionalization. ACS Paragon Plus Environment

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Collectively, the immunofluorescence and AFM images confirmed that M13 bacteriophages were successfully coated on the AgNW surface, rather than forming their own network structure. The AgNWs, BPWHW, BPWHW/AgNW and BPWHW/AgNW formed after centrifugation were further analyzed by FT-IR spectroscopy to verify the adsorption of BPWHW on the AgNW surface. The unbound BPWHW was removed from the BPWHW and AgNW mixture during the centrifugation and redispersion process. As shown in Figure 2c, the spectra of both BPWHW/AgNW and the BPWHW/AgNW after centrifugation showed typical peaks of both AgNWs and BPWHW. The peaks at ~1365 and ~1250 cm-1 were attributed to the CH bending and aromatic CH out-of-plane bending of polyvinylpyrrolidone (PVP) on the AgNW surface, respectively, whereas the peaks at ~2997 and ~1400 cm-1 were attributed to the CH stretching and NH bending vibrations of BPWHW, respectively.18-20 The adhesion of BPWHW on the AgNWs might be induced by the physical adsorption or hydrophobic interaction between the M13 bacteriophage surface proteins and the bare AgNW surface. However, this issue requires further investigation that is beyond the scope of the present work. On the basis of all the results, BPWHW was decorated on the AgNW surface, and BPWHW/AgNW was successfully prepared on GFFP. 3.2. Characterization of genetically modified M13 bacteriophages. The amino acid sequencing and alignment were carried out using the Basic Local Alignment Search Tool (BLAST). The BPWT and BPWHW gene coding base pair sequence was visualized, and the BPWHW sequence had 95 % similarity with that of BPWT (Figure S2a). The amino acids translated from DNA sequences, in which a sequence (AEEWHWQEGD) appeared thrice, were analyzed with the Chromas tool (Technelysium Pty. Ltd.). The DNA sequence of BPWHW showed the WHW peptide insertion at the pVIII site (Figure S2b). Furthermore, the pVIII BPWHW amino acid frequency and identification analysis compared with that of pVIII BPWT is shown in Figure S2c. The WHW sequence shared 85 % identity with the protein M13 f1 pVIII.


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3.3. Raman enhancement and PQ sensitivity test of the BPWHW/AgNW SERS sensor. The SERS effect of the prepared BPWHW/AgNW sensor was tested using a typical Raman reporter dye, R6G, at various concentrations (Figure 3a). The typical R6G peak at 1647 cm-1 was selected for quantification because this peak did not overlap with the background Raman signal of the BPWHW/AgNW. Control Raman spectra of R6G on GFFP and BPWHW-coated GFFP were measured to ensure that the SERS effect was from AgNWs rather than GFFP or M13 bacteriophages (Figure S3a). The molecular concentration on the SERS substrate in this study was described in mole/cm2 or weight/cm2 because the drop volume of the solution was fixed at 20 µL on 1 cm2 of the SERS substrate, and the Raman intensity was measured after complete drying of the solvent. The Raman signal of R6G was measured from 20 to 0.2 pmol/cm2, and it could be observed at concentrations as low as 0.2 pmol/cm2, with a linear range from 10 to 0.2 pmol/cm2 (Figure 3a and 3b). The spectra of R6G at various concentrations on the BPWT/AgNW and AgNW controls exhibited a similar tendency to that of the BPWHW/AgNW (Figure S4). For the sensitivity test of BPWHW/AgNW for the PQ molecule, PQ solutions at concentrations of 100, 50, 25, 10, 5, and 1 ppm were prepared, and 20 µL of each solution was dropped on the BPWHW/AgNW sensor. The typical PQ peak selected was at 1642 cm-1 and could be detected at concentrations as low as 20 ng/cm2, which corresponded to a concentration of 1 ppm in the initial PQ solution (Figure 3c). The PQ standard curve was linear from 20 ng/cm2 to 2000 ng/cm2, with an R2 value of 0.985 (Figure 3d). Additionally, the PQ SERS spectra at various PQ concentrations measured on BPWT/AgNW and AgNW showed a linear relation with the Raman intensities that was similar to that on BPWHW/AgNW (Figure S5). To better understand the sensor sensitivity, the limit of detection (LOD) was calculated using the following formula: LOD = (3 × standard deviation at blank) / Slope of the calibration curve, as reported elsewhere.21 The LOD values of PQ for BPWHW/AgNW, BPWT/AgNW and AgNW were 40.2 ng/cm2, 208 ng/cm2, and 124 ng/cm2, respectively. A comparison of the LODs and linear ranges of SERS sensors developed previously for PQ detection and this work is summarized in Figure S6. However, interestingly, the Raman intensity of PQ on the BPWHW/AgNW 10 ACS Paragon Plus Environment

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sensor was relatively higher than that on BPWT/AgNW and AgNW. In Figure 3e, one example of the PQ Raman intensity at 1 µg/cm2 on each substrate was plotted after baseline adjustment at 1660 cm-1. Further, the Raman spectra of various PQ concentrations (from 2000 ng/cm2 to 20 ng/cm2) on each substrate were compared. At all the PQ concentrations (except 20 ng/cm2), the Raman intensity of PQ on BPWHW/AgNW was statistically higher than that on BPWT/AgNW and AgNW (Figure S7 and 3f). Because of the relatively low signal intensity of PQ on BPWT/AgNW, the LOD of BPWT/AgNW was 5 times and 3 times higher than those of BPWHW/AgNW and AgNW, respectively. Then, the Raman signal reproducibility of BPWHW/AgNW was investigated by calculating the hot spot density in the laser spot area and measuring the Raman intensity deviation of three different PQ concentrations. The Raman laser spot size of the instrument used in this work was 1.68 µm in diameter with an area of 2.22 µm2. Hot spots were counted at AgNW cross points in a 0.25 µm2 area in the SEM image (Figure S8a), and the density was calculated as 355 points/µm2 at least. Due to the high density of hot spots in the laser spot area, we could expect a reproducible Raman signal on the developed BPWHW/AgNW sensor. Further, the BPWHW/AgNW substrates from three different batches were prepared, and the PQ signal at 1647 cm-1 was measured 15 times. As shown in Figure S8b, the Raman intensity deviations of PQ at 1 µg/cm2, 200 ng/cm2 and 100 ng/cm2 were 9.8 %, 11.7 %, and 12 %, respectively. Considering the SD value of 14.8 % from uniform Ag nanorod arrays and the lowest reported SD value of 4.3 % of the SERS sensor,22 the Raman signal of BPWHW/AgNW could be considered reproducible. 3.4. Selectivity comparison of BPWHW/AgNW, BPWT/AgNW and AgNW. For the selectivity test of BPWHW/AgNW, BPWT/AgNW and AgNW, 20 µL of a 50 ppm PQ solution was dropped on each substrate. The Raman signal was measured before and after the washing of each substrate with 10 mL of fresh DI water for 1 min on an orbital shaker at 100 rpm. On the other hand, the optimized BPWHW density on AgNWs was determined after the selectivity test for PQ at several BPWHW concentrations on AgNWs. As shown in Figure S9a, BPWHW at a concentration of 500 µg/mL showed an ununiform BPWHW coating at the boundary region of the GFFP. Additionally, the Raman intensity of PQ was significantly reduced, likely due to the overcoating of BPWHW at hot spots, blocking the 11 ACS Paragon Plus Environment


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approach of PQ. In the case of BPWHW at a concentration of 50 µg/mL, the selectivity for PQ was decreased because of the inefficient coating of BPWHW on the AgNW surface (Figure S9b). The authors prepared BPWHW at a concentration of 100 µg/mL as an optimal BPWHW density in a BPWHW/AgNW SERS sensor. Then, selective PQ detection using the prepared BPWHW/AgNW was compared with that using BPWT/AgNW and AgNW controls. As shown in Figure 4a, BPWHW/AgNW showed a substantial remaining PQ signal after the washing step, while the other substrates showed a significantly reduced PQ signal. The capture efficiency was calculated by subtracting the Raman signal change after the washing step from the initial Raman intensity and then normalizing the result (Figure 4b). In the case of the BPWHW/AgNW surface, 76.4 % of the PQ Raman signal remained, whereas 40.4 % and 36.8 % of the PQ signal was observed in the case of the BPWT/AgNW and AgNW, respectively. The efficiency of PQ captured on BPWHW/AgNW was statistically higher than that on BPWT/AgNW and AgNW. Further, the adhesion and density of PQ on the SERS sensor before and after the washing step were determined from the Raman mapping image obtained from a confocal Raman system within 105 µm × 105 µm. As shown in Figure 4d, BPWHW/AgNW showed significant and uniform selectivity for PQ after the washing step in the entire mapping area compared with BPWT/AgNW and AgNW. Additionally, the Raman intensity changes in the Raman mapping data were well matched with those suggested in Figure 4a. Moreover, the binding capacity of BPWHW and BPWT for PQ was compared by measuring the change in the UV-vis absorbance of PQ before and after centrifugation of the PQ mixture with BPWHW and BPWT. The authors assumed that PQ molecules bound to M13 bacteriophages could be precipitated together with M13 bacteriophages after high-speed centrifugation while the unbound PQ molecules would remain in the supernatant. PQ had a UV-vis absorbance peak at 258 nm and showed a linear relationship over various PQ concentrations (Figure S10). In Figure 4e, the supernatant absorbance of PQ mixed with BPWHW was reduced after high-speed centrifugation compared with that of PQ mixed with BPWT. The adhered weight of PQ to a weight of BPWHW and BPWT was calculated as 6.90 ng/µg and 1.98 ng/µg, respectively. The binding of PQ with BPWT was considered non-specific. ACS Paragon Plus Environment

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3.5. Selectivity tests of BPWHW/AgNW for bipyridylium pesticides. The binding characteristics of BPWHW/AgNW for other bipyridylium pesticides, such as DQ and DIF, were investigated. As shown in Figure S11a and S11b, after the washing step, DQ showed a slightly higher Raman signal on the BPWHW/AgNW substrate than on the BPWT/AgNW and AgNW substrates. On the other hand, DIF showed no significant capture efficiency difference between the BPWHW/AgNW and BPWT/AgNW substrates (Figure S11c and S11d). Among the bipyridylium pesticides, PQ showed the highest binding to BPWHW/AgNW (Figure 4c). The difference in the binding affinity of pesticides to the BPWHW/AgNW surface might result from the different chemical structures, sizes and charge distributions of each pesticide. Furthermore, the PQ selectivity of BPWHW/AgNW in PQ, DQ, and DIF mixtures with 100-fold concentration difference (DQ and DIF at 3000 ppm and PQ at 30 ppm in the initial solution) was tested. As shown in Figure S12, before the washing step, the PQ signal at 1647 cm-1 was not observed due to the dominant Raman signals of DQ and DIF. However, after the washing step, a typical PQ signal (0.6 µg/cm2) in the DQ and DIF (60 µg/cm2) mixture was clearly observed, even with a large concentration difference. This result showed the superior selectivity of BPWHW/AgNW toward PQ even in the presence of DQ and DIF interferences. Considering competitive molecular binding on the sensor surface, we confirmed that the PQ molecule preferentially bound to the BPWHW receptor. As previously reported, the WHW-expressing M13 bacteriophage showed target-specific binding characteristics with trinitrotoluene (TNT) and bisphenol A through the π-π interactions between the WHW sequence and π electrons in TNT and bisphenol A.23-24 Because of the molecular similarities of PQ with TNT and bisphenol A and the presence of π electrons in the structure, PQ was expected to be captured on the BPWHW/AgNW sensor. The authors expected that two tryptophan rings could be bound to the aromatic site of the PQ molecule via π-π stacking. Peptide sequences other than WHW might bind with the PQ molecule, but WHW was one candidate that could be stably expressed on the pVIII domain of the M13 bacteriophage, providing 2 sites of intermolecular π-π interactions. For further characterization of the BPWHW/AgNW selectivity for chemicals that have similar structures to PQ, a selectivity test for cyperquat (CQ), of which half of the chemical structure is similar to that of PQ, was ACS Paragon Plus Environment

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carried out. As shown in Figure S11e, the typical CQ peak at 1630 cm-1 was relatively high after the washing step, with a capture efficiency of 72.3 % on BPWHW/AgNW, compared with that on BPWT/AgNW and AgNW (Figure S11f). CQ showed a similar selectivity tendency as PQ before and after the washing step. Therefore, chemicals with methylated bipyridinium groups in their structure are expected to have a binding affinity for the WHW sequence. Although more than one pesticide could be captured on a the BPWHW/AgNW sensor, the captured pesticides could be identified through analysis of the Raman spectrum of each molecule. We propose two explanations regarding the function of M13 bacteriophages on AgNWs. First, because BPWHW functioned as a receptor for PQ molecules and the filter paper was soaked with target solution from the upper side, the local concentration of PQ on the upper side of the BPWHW/AgNW substrate was increased, enhancing the Raman intensity during the drop-and-dry procedure, whereas the PQ molecules might diffuse throughout the BPWT/AgNW and AgNW substrates, which had less interaction with PQ molecules (Figure S13a). Second, according to the AFM images in Figure 2b, BPWHW could be located at the intersections of AgNWs, which were hot spot areas. The presence of a receptor around hot spots could enhance the Raman signal because the Raman signal intensity was strongly influenced by the distance between the hot spot and target molecule (Figure S13b).25 However, wild-type bacteriophages or M13 bacteriophages that had no specific interaction with the target molecule might act as hindrances for molecules approaching hot spots. Therefore, PQ on BPWT/AgNW showed a relatively low Raman intensity compared with that on BPWHW/AgNW and AgNW. Additionally, for the reasons mentioned above, the DQ and DIF signals were relatively higher on AgNW than on BPWHW/AgNW and BPWT/AgNW, which were considered to have lower binding affinities because they acted as molecular hindrances in hot spot areas. Considering all the results, we conclude that the target molecule, which had a high binding affinity on the BPWHW/AgNW surface, exhibited the highest Raman signal before and after the washing step. 3.6. Field application tests of the BPWHW/AgNW SERS sensor. For the on-site utilization of the developed BPWHW/AgNW SERS sensor, a field application test was conducted using pesticide-treated apples.26-27 The apple surface was vigorously washed with fresh DI water and dried before pesticide 14 ACS Paragon Plus Environment

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treatment. Twenty microliters of PQ solutions at various concentrations was dropped on the surface of an apple and then allowed to dry for 24 h. Then, the BPWHW/AgNW substrate wetted with 20 µL of DI water was wiped on the PQ-treated apple surface 1 min. Because the droplet size of the PQ solution was much smaller than 1 cm2, we reasonably assumed that each dried PQ sample was transferred and spread onto the 1 cm2 BPWHW/AgNW surface during the wiping procedure. After the transfer of each dried PQ sample (from 50 to 1 ppm) on the apple surface to a 1 cm2 BPWHW/AgNW substrate, the density of the transferred PQ residue on the BPWHW/AgNW substrates was calculated as 1.0, 0.5, 0.2, 0.1, and 0.02 µg/cm2, respectively, assuming complete transfer. Figure 5a shows a photograph of the hand-held Raman spectrometer and the BPWHW/AgNW substrate attached to the surface of an apple. As shown in Figure 5b, the transferred PQ signal could be detected at concentrations as low as 0.1 µg/cm2, which is much smaller than the converted PQ MRL density of 31.1 µg/cm2 (PQ MRL in USA, EU, and China: 0.05 mg/kg; the MRL conversion and calculation procedures are presented in the Supporting Information).28 The PQ detection capability of BPWHW/AgNW on an apple surface demonstrated the feasibility of paper-based SERS substrates for on-site residual pesticide detection. Furthermore, BPWHW/AgNW was expected to be useful for selective PQ detection in a complicated matrix, such as ground agricultural products or multiple-pesticide-treated surfaces, by applying a washing procedure. Moreover, by modifying the surface protein sequence of the M13 bacteriophage, the developed M13 bacteriophage-functionalized AgNW sensor could be applied for the detection of various pesticides, chemicals, and biomarkers.


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4. Conclusion In summary, genetically modified M13 bacteriophages were functionalized on the surface of AgNWs and formed a SERS-active network structure for sensitive and selective pesticide detection, especially of PQ molecules. BPWHW/AgNW exhibited substantially enhanced selectivity for PQ compared with BPWT/AgNW and AgNW control SERS substrates. Field application tests of the developed BPWHW/AgNW on PQ-treated apple samples demonstrated the feasibility of using a paper-based SERS sensor for on-site residual pesticide detection. On the basis of this study, the M13 bacteriophagefunctionalized AgNW sensor can be applied in various chemical and biological sensors by genetically modifying the surface peptide sequence of the M13 bacteriophage.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available on the ACS Publication website at DOI:

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] (Dr. Dong-Ho Kim) E-mail: [email protected] (Dr. Ho Sang Jung) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Advanced Production Technology Development Program of the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (316080-04).


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FIGURES

Scheme 1. Schematic illustration of the BPWHW/AgNW sensor structure and pesticide detection on an apple peel surface.


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a)

b) BPWHW/AgNW

GFFP

c)

d)

Figure 1. BPWHW/AgNW surface morphology observed by FE-SEM from the (a) top view and (b) tilting mode view and confocal scanning fluorescence microscopy images of (c) BPWHW/AgNW and (d) AgNW (Scale bar: 20 µm).


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a)

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c) BPWHW/AgNW_Centrifugation BPWHW

Transmittance

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BPWHW/AgNW AgNW

~ 2997 cm-1 ~ 1400 cm-1 ~ 1360 cm-1

~ 1250 cm-1

3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000

800

Wavenumber (cm-1)

Figure 2. AFM images of (a) AgNWs and (b) BPWHW/AgNW and (c) FT-IR spectra of AgNWs, BPWHW, BPWHW/AgNW and BPWHW/AgNW formed after centrifugation.


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a)

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Figure 3. Raman spectra of (a) R6G at various concentrations (b) calibration curve of R6G (c) Raman spectra of PQ solutions at various concentrations (d) calibration curve of PQ (e) Raman spectra of a PQ solution at 1 µg/cm2 on BPWHW/AgNW, BPWT/AgNW, and AgNW and (f) statistical Raman intensity comparison at each concentration (*P < 0.05, **P < 0.01, ***P

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