Optical Fiber-Mediated Immunosensor with a Tunable Detection

Jun 11, 2019 - Last but not least, a signal amplification system is combined with the OFIS to .... After drying under a N2 flow, the bare optical fibe...
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Optical Fiber-Mediated Immunosensor with a Tunable Detection Range for Multiplexed Analysis of Veterinary Drug Residues Rongbin Nie,† Xuexue Xu,† Yiping Chen,*,‡ and Li Yang*,† †

Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, PR China ‡ College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China

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S Supporting Information *

ABSTRACT: We describe herein a newly developed chemiluminescent optical fiber immunosensor (OFIS) with a tunable detection range for multiplexed analysis of veterinary drug residues with vastly different concentrations in milk samples. The optical fiber probe is used as a carrier of biorecognition element as well as a transducer, enabling a low-cost compact design, which makes this system suitable for cost-effective on-site detection of the target analytes. Importantly, the synergy between modulation of the length of the optical fiber sensing region and the number of fibers allows performing multiplexed immunoassays in an easily controllable manner over a tunable detection range from pg/mL to μg/mL analyte concentrations. By combining the optical fiber sensor with a nanocomplex signal amplification system, a highly sensitive chemiluminescent OFIS system is demonstrated for the multiplexed assaying of veterinary drug residues in milk samples with linear ranges of 10−(2 × 104) pg/mL for chloramphenicol, 0.5−500 ng/mL for sulfadiazine, and 0.1−300 μg/mL for neomycin. This controllable strategy, based on modulation of the fiber probe, provides a versatile platform for multiplexed quantitative detection of both low-abundance and high-abundance targets, which shows great potential for on-site testing in food safety. KEYWORDS: optical fiber immunosensor, tunable detection range, signal amplification, veterinary drug residues, multiplexed analysis, on-site detection linked immunosorbent assays (ELISA),11,12 lateral flow immunoassays (LFA),13−17 nanosensors based on molecular recognition,18 and instrumental analyses.19,20 However, these current techniques face several issues, including limited detection ranges, dependence on bulk instrumentation, and being time/labor-consuming. To meet the requirements for the sensitive detection of multiple veterinary drugs, it is of vital importance to develop a reliable sensor for multiplexed assays with a tunable detection range, which is capable of on-site screening and detection of trace amounts of veterinary drug residues in mass-produced food products. The use of a chemiluminescent optical fiber immunosensor (OFIS) is a promising approach due to its ability to detect a broad range of analytes with high sensitivity and specificity.21−25 This technique is based on the traditional ELISA method and uses horseradish peroxidase (HRP) to generate a chemiluminescent signal. Optical fibers are ideal transducers for chemiluminescent signals, and their silica composition enables the efficient immobilization of various biospecific recognition molecules (enzymes, immunomolecules, microorganisms, etc.) via well-known silicon chemistry.26 OFISs offer advantages in regard to the miniaturization and

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ith the rapid development of animal culture, a large number of chemical compounds have been used as veterinary drugs for therapeutic or diagnostic purposes.1−3 The abuse of veterinary drugs results in many problems; thus, both the World Health Organization (WHO) and Food and Agriculture Organization (FAO) have established minimum required performance levels (MRPLs) for forbidden substances regarding veterinary drug residues and maximum residue limits (MRLs) for authorized drugs.4,5 Simultaneous and multiplexed assays of different targets are of essential importance and can greatly enhance the accuracy and reduce the amount of reagents and antibiotics needed for assays.6−9 The main challenge for multiplexed detection of veterinary drugs is that the concentrations of different targets can vary greatly from pg/mL to μg/mL in food products. For instance, as a representative of the broad-spectrum antibiotics widely used in veterinary medicine, chloramphenicol has an MRPL of 300 pg/mL in milk-based foods, while the MRLs for sulfadiazine and neomycin are 100 ng/mL and 1.5 μg/mL, respectively.10 This requires multiplexed analysis of veterinary drugs to be performed over a broad range of concentrations; thus, an analytical platform that possesses a sufficiently broad linear range and high detection sensitivity is in high demand. Efforts have been made in the development of analytical methods for assaying multiple veterinary drug residues in food samples. These methods can be broadly classified as enzyme© XXXX American Chemical Society

Received: April 5, 2019 Accepted: June 11, 2019 Published: June 11, 2019 A

DOI: 10.1021/acssensors.9b00653 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors Scheme 1. Process for Preparing the Sensing Region of Each Bundle of the OFISa

Amine groups are introduced to the silica surface of each bare fiber core by a 3-ADMS solution. BSA conjugates are covalently bonded to the fiber surface in the recognition sensing region in the presence of linking regents EDC/NHS. GSSG is used to block the residue sites to reduce nonspecific adsorption. By bundling multiple fibers and adjusting both the length of the sensing region and the number of bare fiber cores per bundle, the OFIS can achieve multiplexed assaying with a tunable detection range from pg/mL to μg/mL for different veterinary drugs. a

detection sensitivity of the OFIS immunoassays by applying these streptavidin−biotin−HRP nanocomplexes. Thus, the proposed OFIS offers bioassays over an adjustable linear range and ultrahigh sensitivity. The performance of the OFIS is demonstrated by analyzing different levels (a broad range from pg/mL to μg/mL) of three veterinary drug residues (chloramphenicol, sulfadiazine, and neomycin) in milk samples. The limit of detection (LOD), linear detection range, specificity, and recovery of the proposed OFIS for the detection of chloramphenicol, sulfadiazine, and neomycin are systemically evaluated. Our study should greatly increase the efficiency of the assays of veterinary drug residues in massproduced food products.

portability of analytical devices; hence, OFISs have emerged as a promising candidate for field detection.27,28 However, it is still difficult for current OFIS bioassays to achieve sensitive detection of multiple targets over a tunable and broad linear range due to the limited immobilization capacity of a micronscale optical fiber core and the uncontrollable immobilization of the active proteins for the desired dynamic detection range. Here, we propose a new OFIS that is capable of performing ultrasensitive and quantitative assays with tunable detection ranges for the multiplexed detection of veterinary drugs (Scheme 1 and Scheme 2). The proposed OFIS assay contains several essential novel strategies that can overcome the aforementioned difficulties of normal OFIS bioassays. First, both the length and the number of bare fiber cores can be adjusted in this OFIS assays, leading to the controllable and quantitative immobilization of antigens for different target samples. To the best of our knowledge, it is the first time that an adjustable linear range has been achieved via controllable modulation of fibers. Moreover, gathering multiple fibers into a bundle serves to further amplify the signal readout in a controllable manner. Second, we utilize a nonliquid-flow system for the whole assay process. Thus, the method is very suitable for on-site detection of veterinary drug residues and greatly simplifies the immunosensor device and reduces the cost. Last but not least, a signal amplification system is combined with the OFIS to amplify the chemiluminescent signal readout, which is based on HRP-catalyzed reactions. In a previous study, we reported a signal amplification system using the one-step self-assembly of streptavidin and biotin-HRP to form a streptavidin−biotin−HRP nanocomplex that can be combined with microfluidic assays to sensitively detect two biomarkers of infectious diseases.29 With the aid of its extremely high binding affinity, we can greatly improve the

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EXPERIMENTAL SECTION

Reagents and Apparatus. Three veterinary drugs (chloramphenicol, sulfadiazine, and neomycin), hapten-carrier protein conjugates (chloramphenicol-BSA (1 mg/mL), sulfadiazine-BSA (1 mg/mL), and neomycin-BSA (1 mg/mL)) and antibodies (1 mg/mL) were obtained from Lufeifan Biology (Henan, China). Sulfo-NHS-LCBiotin (Biotin) was obtained from ApexBio (Texas, USA). 3Aminopropyl diethoxymethylsilane (3-ADMS), 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide hydrochloride (EDC), Nhydroxy-succinimide (NHS), o-Dianisidine (ODA), and oxidized glutathione (GSSG) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Biotinylated horseradish peroxidase (biotinHRP) was purchased from Bioss Biotechnology (Beijing, China). Streptavidin and chemiluminescent substrate reagent kits were obtained from Solarbio Life Sciences (Beijing, China). Hydrofluoric acid (48%, w/w) was obtained from Aladdin (Shanghai, China). Dimethylformamide (DMF), CHCl3, and NaHCO3 were obtained from Beijing Chem Works (Beijing, China). Na2HPO4 and NaH2PO4, which were used to prepare 10 mM solutions of phosphate buffered saline (PBS, pH 7.4), were purchased from Tianjin Guangfu Technology Development Co., Ltd. (Tianjin, China). The water B

DOI: 10.1021/acssensors.9b00653 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors Scheme 2. Multiplexed Assay of Veterinary Drugs Using the Proposed OFISa

a

The overall setup of the assay is presented in the top-left panel. The multiplex assay is accomplished by transferring the OFIS from the sample vial (I) to an array of vials (II)-(V), as shown in the lower-left panel. (I) Sample vial containing a mixture of the three drugs and their corresponding BAbs for specific immunoreactions; (III) vials containing the streptavidin-biotin-HRP nanocomplex solution to introduce the signal amplification system to each bundle of the OFIS; (V) chemiluminescent (CL) detection of each bundle of the OFIS. Schematic diagrams of steps (I), (III), and (V) are presented in the right panel. Steps (II) and (IV) contain PBST buffer for washing the bundles between steps. See text for details. streptavidin solution and the biotin−HRP solution for 30 min at room temperature. Surface Modification of the Optical Fiber for OFIS. For the proposed OFIS, three bundles of optical fibers were gathered together for the multiplexed assays of veterinary drugs over a broad range of concentrations. Each bundle of the OFIS was designed for the specific immunoassay of one drug target. Scheme 1 shows the process for preparing the sensing region on the fiber surface of each bundle. Optical fibers with a numerical aperture (NA) of 0.22 (SFS400/440/ 700T Superguide UV−vis, Wyoptics Technology Co., Ltd. Shanghai, China) were used to prepare the OFIS. The fiber core was 400 μm in diameter (refractive index of 1.457 at 633 nm) and surrounded by a 20 μm silica cladding (refractive index of 1.439 at 633 nm) and a 130μm-thick acrylic-jacket layer. The total length of each optical fiber used for the OFIS assay was 20 cm, with the acrylic-jacket layer removed from its distal end for immobilization of the biorecognition

used in the experiments was deionized by a water purification system (Thermos Fisher, USA). Preparation of Biotin-Ab and Streptavidin−Biotin−HRP Nanocomplex. The biotinylated antibodies (B-Ab of chloramphenicol, sulfadiazine, and neomycin) and streptavidin−biotin−HRP conjugates were prepared according to the method described in a previous study with some modifications.29 Sulfo-NHS-LC-Biotin (Biotin) was diluted to 1 mg/mL with DMF, and Ab was diluted to 1 mg/mL with a 0.1 g/L NaHCO3 solution (pH 9.6). The solutions were mixed in a molar ratio of 20:1 (Biotin:Ab) and stirred for 1 h at 37 °C. Then, the mixed solution was centrifuged with a centrifugal ultrafiltration unit (10 kDa filter) at 9000 rpm for 10 min at 4 °C to remove excess biotin and ions. After washing 3 times using PBS (10 mM, pH 7), the solution was diluted at −20 °C. The solution of the streptavidin−biotin−HRP conjugate was obtained by mixing the C

DOI: 10.1021/acssensors.9b00653 ACS Sens. XXXX, XXX, XXX−XXX

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Figure 1. Characterization of the immobilized hapten-carrier BSA-conjugates on the fiber surface by (A) UV−vis spectroscopy and (B) FTIR spectroscopy of a bare fiber (black line), a BSA-coated fiber (blue line), and the free BSA protein (red line). molecules to form the sensing region. To remove the acrylic-jacket layer, the distal end of the optical fiber was inserted into chloroform for 10 min at the desired immersion length. Afterward, the acrylic jacket was carefully stripped with a knife. The silica cladding of the fiber was removed by chemically etching in hydrofluoric acid (24%) for 20 min, followed by rinsing with 0.1 M NaOH and water 3 times each. After drying under a N2 flow, the bare optical fiber core was ready for subsequent silanization and modification with the biorecognition molecules. Hapten-carrier protein conjugates (BSA-chloramphenicol, BSAsulfadiazine, or BSA-neomycin) were immobilized onto the fiber core to prepare the OFIS. First, the hydroxyl groups on the SiO2 surface of the recognition sensing region of the fiber were activated by a NaOH solution, and then the bare optical fiber core was immersed in a 1% (v/v) 3-ADMS solution in ethanol for 1 h to introduce primary amine groups onto the silica surface. After washing with ethanol 3 times and drying under a N2 flow, the fiber core was immersed in a phosphate buffer solution (0.1 mM, pH 7.0) containing 0.015 M EDC and 0.03 M NHS for 1.5 h and then immediately inserted into the haptencarrier protein conjugate solutions (BSA-chloramphenicol, BSAsulfadiazine, or BSA-neomycin) for another 1.5 h. The free carboxylic groups on the BSA can be converted into reactive intermediates in the presence of the linking regents EDC/NHS, which are susceptible to attack by the amine groups of the fiber core, forming covalent bonds between the BSA and the fiber surface in the recognition sensing region. After washing away the unbound hapten-carrier protein conjugates with PBS, a 10 mg/mL solution of GSSG in PBS was used for 2 h to block the residue sites to reduce nonspecific adsorption. After washing with PBST, the fibers with the desired recognition sensing lengths (sensing region) were assembled into bundles with different numbers of fibers and then fixed with a shrinkable sleeve. The prepared OFISs were kept at 4 °C when not in use. Multiplexed Detection of Veterinary Drugs Using the Chemiluminescent OFIS. A schematic diagram of the chemiluminescent OFIS for multiplexed detection of veterinary drugs is shown in Scheme 2. The assay used in the present study was based on an indirect competitive immunoassay. Briefly, the OFIS with immobilized hapten-carrier protein conjugates (i.e., BSA-chloramphenicol, BSA-sulfadiazine, or BSA-neomycin) was regarded as the biorecognition element. Samples containing the different veterinary drugs were premixed with their corresponding B-Abs, allowing a portion of the antibodies to be specifically bound. The occupied antibody binding sites were proportional to the concentration of the corresponding veterinary drug in the sample. After immersing the sensing region of the optical fiber probe into the mixture, the remaining free B-Abs binding sites would bind to the hapten-carrier protein conjugates immobilized on the OFIS. Finally, chemiluminescent detection was employed for signal readout of the sensor.

To achieve a multiplexed assay, our OFIS was uniquely designed by combining three bundles of fiber cores together. For each bundle, the surface of the sensing region was immobilized with hapten-carrier protein conjugates of one veterinary drug, and the sensing length and number of fiber cores were adjustable in consideration of the detection sensitivity of the corresponding target. A specific immunoreaction occurred at each bundle of the OFIS as the sensor was immersed in the prereacted mixtures (the samples containing three drugs and their corresponding B-Abs). After incubation for 1 h at 37 °C, the OFIS was washed with PBST three times to remove the excess veterinary drugs and B-Abs and then transferred to three individual vials containing the streptavidin−biotin−HRP nanocomplex solution. Thus, the streptavidin−biotin−HRP nanocomplex-signal amplification system, which can significantly enhance the detection sensitivity, was introduced to each bunch for the specific detection of the target drug. The large concentration of HRP in the nanocomplex can catalyze the decomposition of H2O2 into H2O and free-radical oxygen, which oxidize luminol to generate luminescence. After incubation for 30 min at 37 °C and washing with PBST three times, the recognition sensing regions of the bundled fibers were then immersed in chemiluminescent (CL) substrates (composed of H2O2 and luminol) to generate the chemiluminescent signals. A photon counting detector (CH326, Beijing Hamamatsu Photon Technique INC) was used to detect the chemiluminescent signal by directly placing the end of the OFIS against the detector window, thus avoiding the need for any other optical elements. Traditional ELISA Procedure. For traditional ELISA procedure, 96-well plates were coated with hapten-carrier protein conjugate (BSA-chloramphenicol) by adding 100 μL of the conjugate in PBS per well and incubating overnight at 4 °C. After removing away the unbound hapten-carrier protein conjugates with PBST and blocking the residue sites with 10 mg/mL GSSG in PBS for 2 h, the plates were washed with PBST for 3 times, then treated with the mixed solutions of 50 μL HRP labeled antibodies of chloramphenicol and 50 μL chloramphenicol in PBS with different concentrations. After incubating for 2 h at 37 °C, the plates were washed with PBST thoroughly. Then, 100 μL of substrate solution (10 mM ODA and 0.01% H2O2 in PBS) was added to each well. After 15 min incubation, the absorbance at 460 nm was measured by 96-well plate reader (Infinite M Plex, TECAN, CH). Spiking Tests of Chloramphenicol, Sulfadiazine, or Neomycin in Milk Samples. For the spiking tests, three milk samples purchased from the local supermarket were spiked with chloramphenicol, sulfadiazine, or neomycin at different levels. After ultrafiltration−centrifugation, the ultrafiltrate was collected and diluted 10fold with PBS prior to detection. In the spiked samples, the final concentrations of chloramphenicol, sulfadiazine, or neomycin were 0 pg/mL, 20 pg/mL, 200 pg/mL, and 2000 pg/mL chloramphenicol; 0 ng/mL, 2 ng/mL, 20 ng/mL, and 200 ng/mLsulfadiazine; and 0 μg/ D

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Figure 2. Semilogarithmic plots of the response of the OFIS for the multiplexed analysis of chloramphenicol, sulfadiazine, and neomycin based on changes in the length of the recognition sensing region (A1−A3) and the number of fiber cores (B1−B3) of each bundle. mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL neomycin. All tests were performed three times to obtain precise results.

indicating the maximum immobilization efficiency of the conjugates on the sensing region of each bundle. Accordingly, for the OFIS prepared under the above optimal concentrations of the BSA-conjugates, the concentrations of B-Ab for each drug sample was optimized to obtain a complete immunoreaction with the corresponding conjugates, for which the concentrations are 5 μg/mL for chloramphenicol, 10 μg/mL for sulfadiazine, and 40 μg/mL for neomycin (see Figure S1 (A2, B2, and C2)). The immunoreaction time between B-Ab and the BSA-conjugates was investigated over the range of 5 to 75 min for each drug sample. As shown in Figure S1 (A3, B3, and C3), the chemiluminescent intensity increases until plateauing when the immunoreaction time reaches 60 min for chloramphenicol, sulfadiazine, and neomycin, indicating that the reaction is complete. An immune reaction time of 60 min is used in the OFIS assay considering the multiplexed detection of the three drug residues. One feature of the proposed method is the combination of a streptavidin−biotin−HRP nanocomplex-signal amplification system with chemiluminescent detection, which is expected to enhance the sensitivity for assaying veterinary drug residues. Taking the detection of chloramphenicol as an example, we investigated and optimized two important factors, i.e., the molar ratios of streptavidin to the biotin-HRP and streptavidin concentrations, in the signal amplification system, as shown in Figure S2 (A and B), respectively. Keeping the concentration of biotin-HRP at 12.5 μg/mL, we achieved the maximal chemiluminescent signal at a molar ratio of streptavidin to biotin-HRP of 1:4 (Figure S2 (A)). Further increasing the molar ratio results in a decrease in the chemiluminescent signal due to the high concentration of streptavidin, which may lead to an incomplete reaction between the streptavidin and biotinHRP. With a molar ratio of streptavidin to biotin-HRP of 1:4, the maximum chemiluminescent signal was obtained at 1.25 μg/mL streptavidin (Figure S2 (B)). While a low concentration of streptavidin is insufficient to generate a high chemiluminescent intensity, the signal would be degraded at high streptavidin concentrations due to steric hindrance. Multiplexed Detection of Veterinary Drugs Using OFIS. As aforementioned, multiplexed analysis of veterinary drug residues can be accomplished based on the specific immunoreaction that occurs at the sensing region of each

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RESULTS AND DISCUSSION Characterization and Optimization of Immunoassay. It is difficult to directly immobilize veterinary drugs onto the optical fiber surface because veterinary drugs have low molecular weights; thus, hapten-carrier protein conjugates (BSA-chloramphenicol, BSA-sulfadiazine, or BSA-neomycin) were immobilized onto the optical fiber surface to prepare the OFISs. Both UV−vis and FTIR spectroscopy were employed to ensure the successful immobilization of the hapten-carrier BSA conjugates on the fiber surface, which is essential for the indirect competitive immunoassay using the proposed OFIS. Figure 1A,B shows the measured UV and FTIR spectra, respectively, for a fiber modified by the hapten-carrier protein conjugates, a bare fiber, and the free BSA protein. The characteristic absorption peak of the proteins appears at 280 nm in the UV spectra of BSA and the conjugate-modified fiber. In the FTIR spectrum (Figure 1B), the two peaks at 1658 and 1527 cm−1 for the BSA protein can be assigned to the CO stretching and the NH bending of BSA. These characteristic protein peaks are clearly observed in the FTIR spectrum of the conjugate-modified fiber. In addition, the peak at 1849 cm−1, which is not observed in the spectrum of the free BSA protein, can be assigned to the CO stretching mode of the linking reagents EDC/NHS. Both the UV and FTIR spectra strongly indicate that the hapten-carrier protein conjugates are immobilized onto the recognition sensing region of the fiber. We investigated several essential factors that may affect the immunoassay of veterinary drugs using the proposed OFIS. For all the experiments in this section, the sample solutions only contain the B-Abs of the drugs. Thus, an immune reaction occurs between the B-Abs in solution and hapten-carrier protein conjugates immobilized on the fibers, and the chemiluminescence intensity (I0) of each fiber bundle of the OFIS is measured. We first measured the intensity I0 for several different concentrations of the hapten-carrier protein conjugates. As shown in Figure S1 (A1, B1, and C1), for the assay of each drug residue, the maximum chemiluminescent intensity is observed at 10 μg/mL BSA-chloramphenicol, at 20 μg/mL BSA-sulfadiazine, or at 80 μg/mL BSA-neomycin, E

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Figure 3. (A) Linear range of the OFIS for the multiplexed detection of chloramphenicol, sulfonamides, and neomycin. (B) Effect of signal amplification on the sensitivity of the OFIS for the detection of chloramphenicol based on the chemiluminescent intensity for the detection of chloramphenicol with/without the signal amplification system.

region and the same number of fibers, the response ranges of the sensors for the three veterinary drugs are different. However, such a difference in linear ranges caused by the nature of the antibodies does not meet the analytical requirements of targets with broadly ranged concentrations. For example, using one fiber with 0.5 cm sensing region, the linear ranges of the sensors for chloramphenicol and neomycin is 10−20 000 pg/mL and 0.01−0.1 μg/mL, respectively (black lines in Figure 2 A1 and A3). The result is acceptable for the analysis of chloramphenicol, which has an MRPL of 300 pg/ mL in milk; but it is not sufficient for neomycin, whose MRL is 1.5 μg/mL in milk. By changing the length of fiber sensing region to 1.5 cm and the number of fibers to 8, the linear range for neomycin is adjusted to 0.1−300 μg/mL, which is broad enough for the analysis of neomycin. Therefore, even the nature of the antibodies has an effect on the linear range, the modulation of fiber is no doubt necessary to analyze targets with broadly ranged concentrations. Considering the remarkably different MRPLs of the veterinary drug residues in the milk samples in the present study, we assembled 0.5 cm, 1 fiber core; 1.0 cm, 5 fiber core; and 1.5 cm, 8 fiber core bundles for each of the three bundles of the OFIS for assaying chloramphenicol, sulfadiazine, and neomycin. Thus, multiplexed immunoassays of the veterinary drugs can be accomplished using an OFIS over detection ranges of 10−20 000 pg/mL for chloramphenicol, 0.5−500 ng/mL for sulfadiazine, and 0.1−300 μg/mL for neomycin (Figure 3A). In other words, our method enables, on one hand, the highly sensitive detection of chloramphenicol at the pg/mL level, and on the other hand, a broad enough detection range to detect sulfadiazine and neomycin in the ng/mL to μg/mL range. This feature of the proposed OFIS can meet the requirement for multiplexed detection of veterinary drug residues whose concentrations are widely different in food products. For quantitative detection, the linear relationships of In vs log[C] are determined to be In = 0.2643 log[C] − 0.1860 (R2 = 0.9889) for chloramphenicol, In = 0.2028 log[C] − 0.2199 (R2 = 0.9719) for sulfadiazine, and In = 0.1409 log[C] − 0.1906 (R2 = 0.9782) for neomycin. These results show that modulating the sensing region length and the number of optical fibers is a straightforward and effective strategy to adjust the detection range of an immunosensor to meet the requirements for multiplexed analysis of multiple targets with different concentrations.

bundle of the proposed OFIS system. In this section, we show the feasibility of the proposed OFIS for the multiplexed immunoassay of veterinary drugs over tunable detection ranges. For competitive immunoassays, the response of the OFIS toward a target is defined as the normalized chemiluminescent signal intensity (In) In = (I0 − It )/I0

where I0 is the intensity of the net chemiluminescent signal of the solution without the target, and It is that of the sample containing the target. In Figure 2 (A1, A2, and A3), the response of the OFIS with changing lengths of the recognition sensing region of each bundle is shown. In these multiplexed assays, only one fiber core composed each bundle of the OFIS. A good linear relation of the response In vs log[C] is obtained for each veterinary drug (here, C denotes the sample concentration). One can see that by changing the length of the recognition sensing region from 0.5 to 1.5 cm, the detection range shifts to nearly one order-of-magnitude higher concentrations for either of the three samples. In Figure 2 (B1, B2, and B3), we show the effect of the fiber number on the response of the OFIS for each veterinary drug. For these assays, we prepared OFISs with one, five, or eight fiber cores for each bundle while keeping the lengths of the sensing regions to 0.5 cm for the chloramphenicol bundle, 1.0 cm for the sulfadiazine bundle, and 1.5 cm for the neomycin bundle. Again, for each sample, the detection range shifts to higher concentrations with increasing numbers of fiber cores. The results in Figure 2 indicate that by changing the number of fiber cores and the lengths of the recognition sensing regions of each bundle of the OFIS, the linear range for assaying the veterinary drugs can be adjusted between the pg/mL and μg/ mL ranges, which can be attributed to the adjustable amount of hapten-carrier protein conjugates immobilized on the sensing region of each bundle. Thus, using the proposed OFIS, one can perform multiplexed assays in which a broad detection range is required for different samples. Note that the limit-of-detection of the OFIS also shifts to higher concentrations for either a longer sensing region or a higher number of fiber cores. It should be mentioned that different antibodies may result in different dynamic working ranges due to their nature and antibody−target recognition mechanisms. This could be seen in Figure 2 (A1-A3): with the same length of the fiber sensing F

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ACS Sensors We further investigated the responses of the sensor for each drug with and without a streptavidin−biotin−HRP nanocomplex-signal amplification system. A significant enhancement of the response is observed by using the signal amplification system (see Figure 3B for the results of chloramphenicol and Figure S3 in Supporting Information for those of the other two drugs). The limits of detection (LOD) with the signal amplification system, which is calculated by 3S/M (where S is the value of the standard deviation of the baseline of the blank and M is the slope of the standard calibration curve), are determined to be 2.86 pg/mL, 0.22 ng/mL, and 0.03 μg/mL for chloramphenicol, sulfadiazine, and neomycin, respectively, which is approximately 70− 230 times lower than that without a signal amplification system. In Table S1, we compare the sensitivity and detection range of the proposed OFIS for the detection of veterinary drugs with those obtained using other analytical platforms that have been reported in the literature. Compared to those of other analytical platforms, our method shows satisfactory LODs and a linear detection range. Moreover, the proposed OFIS exhibits several unique merits. In addition to enabling multiplexed assaying over a broad detection range and the combination of a signal amplification system for enhancing the detection sensitivity, which we have discussed above, the OFIS is compactly designed and has no liquid-flow systems or bulk equipment. That is, all the steps involved in the competitive immunoassay, including the binding of the targets in the samples, washing, and subsequent detection are accomplished by immersing the sensing region of the probe into each microvial in the reagent disk. Thus, the biosensor device is greatly simplified, making it suitable and portable for on-site detection. We also compare the assays using the proposed OFIS to those using the traditional ELISA method. Taking chloramphenicol as an example, the results show that the linear range, sensitivity and analysis speed of the OFIS are greatly improved comparing to the conventional ELISA. The LOD of the OFIS is nearly 500 times lower than that of the conventional ELISA (2.86 pg/mL for OFIS and 1429 pg/mL for ELISA) with the wider detection range (10−(2 × 104) pg/mL for OFIS and 5000−(8 × 104) pg/mL for ELISA); meanwhile, the analysis speed is greatly improved (1.5 h for OFIS and 2.5 h for ELISA). Moreover, OFIS is multiplex mode with tunable detection range, which can provide more information, decrease cost, and improve detection efficiency. Specificity of the OFIS for Multiplexed Detection of Veterinary Drugs. To achieve accurate multiplexed assays using the OFIS, it is essential to ensure that there is little interference between different samples. Thus, we investigated the specificity of the sensor by assaying one veterinary drug with the other two drugs as interferons. The concentrations of chloramphenicol, sulfadiazine and neomycin used in the interference study were 1000 pg/mL, 20 ng/mL, and 100 μg/mL, respectively. The results are shown in Figure 4, in which the response of the sensor is normalized to that of the target drug without interferons. Clearly, there is negligible interference from the other drugs on the assay of the target veterinary drug. We also evaluated the stability and variability of the OFISs toward the detection of chloramphenicol (concentration range of 10−20 000 pg/mL), sulfadiazine (concentration range of 0.5−500 ng/mL), and neomycin (concentration range of 0.1−300 μg/mL). The intracoefficient

Figure 4. Specificity of the OFIS for the detection of the three veterinary drugs. The relative ratio represents the percentage of the target found in the presence of the other drugs, which acted as interferons. The error bars are the standard deviations calculated from three measurements (n = 3).

of variation (CV) and the inter-CV are below 9% and 12%, respectively (Table S2). It should be noted that, while the presented strategy includes several processes of fiber modification, nanocomplex synthesis, etc., the good intraand interassay coefficients of variation of the analysis results G

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ACS Sensors Table 1. Determination of Chloramphenicol, Sulfadiazine, and Neomycin in Spiked Milk Samplesa Chloramphenicol (pg/mL)

Sulfadiazine (ng/mL)

Neomycin (μg/mL)

Sample

Spiked

20

200

2000

2

20

200

1

10

100

1

Found Recovery% RSD%(n = 3) Found Recovery% RSD%(n = 3) Found Recovery% RSD%(n = 3)

17.94 89.7 6.33 18.70 93.5 5.11 18.45 92.2 5.22

201.39 100.7 7.36 193.89 96.9 3.93 187.32 93.7 1.88

2168.54 108.4 4.82 2045.04 102.2 1.21 2017.02 100.9 4.95

1.90 95.0 3.42 2.01 100.5 3.52 1.86 93.0 3.62

19.91 99.6 1.56 17.84 89.2 1.59 20.82 100.4 4.72

174.46 87.3 3.39 208.75 104.4 3.15 180.53 90.3 3.62

1.04 104.0 2.40 0.91 91.0 4.15 0.85 85.0 2.56

10.30 103.0 5.92 10.94 109.4 11.28 10.63 106.3 3.67

109.20 109.2 7.14 101.44 101.4 4.89 92.32 92.3 8.13

2

3

a

Each value is the mean of three determinations.

showed that the stability and reproducibility of the method are satisfactory. Multiplexed Assay of Veterinary Drugs in Milk Samples. To verify the feasibility and accuracy of the proposed method, the designed OFIS was utilized for multiplexed assaying of chloramphenicol, sulfadiazine, and neomycin in milk samples that contain various proteins and other interferons. Different concentrations of chloramphenicol, sulfadiazine, and neomycin were spiked into three milk samples. The high recoveries for the detection of chloramphenicol (89.7−108.4%), sulfadiazine (87.3−104.4%) and neomycin (85.0−109.4%) suggest that this approach works well in milk samples (Table 1). The relative standard deviations (RSDs) are below 12%, indicating good reproducibility of this method. The results confirm that the proposed OFIS is highly practical and reliable for the determination of multiple veterinary drug residues in complex samples.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yiping Chen: 0000-0001-9309-2730 Li Yang: 0000-0001-6723-7377 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grant nos. 21775017, 21475019, 81671784) and the Natural Science Foundation of Jilin Province, China (Grant No. 20180101174JC). L. Yang would also like to thank the support from Jilin Provincial Department of Education and Jilin Provincial Key Laboratory of Micro-Nano Functional Materials (Northeast Normal University).

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CONCLUSIONS In summary, we report a novel chemiluminescent OFIS for multiplexed assaying of veterinary drug residues with amplified sensitivity and a tunable detection range. Modulating the length of the sensing region and the number of optical fibers allows the multiplexed detection of chloramphenicol, sulfadiazine, and neomycin over a broad detection range from pg/mL to μg/mL. Combined with a nonliquid-flow system, the OFIS can realize multiplexed detection with good operability. A streptavidin−biotin−HRP amplification system is employed to provide a significantly enhanced sensitivity for quantitative detection. Three veterinary drug residues spiked into real milk samples are quantitatively detected via the proposed immunosensor with good reproducibility. This technique provides a new multiplexed platform for simultaneous and quantitative analysis of both low-abundance and highabundance veterinary drug residues over a broad detection range, which has great promise for application in food analysis and food safety control. Further work will focus on developing a highly sensitive OFIS assay with a broad detection range for molecular diagnosis.

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comparison of the analytical performances, investigation of stability and repeatability (PDF)

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00653. Optimization of the experimental conditions, comparison of responses with/without signal amplification, H

DOI: 10.1021/acssensors.9b00653 ACS Sens. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acssensors.9b00653 ACS Sens. XXXX, XXX, XXX−XXX