Microchip electrophoresis array-based aptasensor for multiplex antibiotic detection using functionalized magnetic beads and polymerase chain reaction amplification

Accepted Manuscript Title: Microchip electrophoresis array-based aptasensor for multiplex antibiotic detection using functionalized magnetic beads and polymerase chain reaction amplification Authors: Lingying Zhou, Ning Gan, Futao Hu, Tianhua Li, Yuting Cao, Dazhen Wu PII: DOI: Reference:

S0925-4005(18)30415-5 https://doi.org/10.1016/j.snb.2018.02.136 SNB 24231

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

13-8-2017 30-1-2018 18-2-2018

Please cite this article as: Lingying Zhou, Ning Gan, Futao Hu, Tianhua Li, Yuting Cao, Dazhen Wu, Microchip electrophoresis array-based aptasensor for multiplex antibiotic detection using functionalized magnetic beads and polymerase chain reaction amplification, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.02.136 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Microchip electrophoresis array-based aptasensor for multiplex antibiotic detection using functionalized magnetic beads and

Lingying Zhoua, Ning Gan*,a, Futao Hub, Tianhua Lia, Yuting Caoa, Dazhen Wua

of Material Science and Chemical Engineering, Ningbo University, Ningbo, China,

31521 bFaculty

of Marine, Ningbo University, Ningbo, China, 31521

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a Faculty

*Corresponding Author Tel: +86-574-87609987; Fax: +86-574-8760998

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E-mail address: [email protected]

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Graphical abstract

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polymerase chain reaction amplification

Highlights An MCE array-based aptasensor to detect multiplex antibiotics was developed.

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The target can be directly detected by MCE without any derivatization。

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The detection throughput obviously increased to at most 48 samples in one hour

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It is a universal detection platform for different kinds of antibiotics.

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High sensitivity to antibiotics with LOD of 0.0025 nM was obtained by PCR.

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Abstract：A microchip electrophoresis (MCE) array-based aptasensor for multiplex

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detection of antibiotics was developed using multi-capture DNA functionalized magnetic beads (AuMPs@captureDNA@assistantDNA) and polymerase chain reaction (PCR) for signal amplification. In this study, kanamycin (KANA) and

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chloramphenicol (CAP) were employed as model analytes. In the presence of a target,

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specific binding between the target and corresponding capture DNA (C-DNA) would

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induce the unwinding of double-strand structures, leading to the release of assistant DNA (A-DNA) into the supernatant after magnetic separation. The released A-DNA

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was co-amplified by PCR with an internal standard strand (I-DNA). After 20 cycles of

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PCR, the ratio (IA-DNA/II-DNA) between the above PCR products was proportional to the concentrations of the target with a detection limit of 0.0025 nM and 0.006 nM for KANA and CAP, respectively. Under optimal conditions, the method exhibited high

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sensitivity and selectivity for the targets, and the average detection time was about 1 min. Moreover, this assay was successfully employed to detect KANA and CAP in

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milk and fish samples with consistent results to that of enzyme-linked immunosorbent assay, suggesting that it is a promising platform for detecting small molecules in food

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by changing the corresponding aptamer.

Keywords: microchip electrophoresis array, aptamer functionalized magnetic beads, antibiotics detection, PCR, food 1. Introduction Recently, achieving multiplex detection of antibiotic residues in food has become a priority because of the strong side effects of antibiotics on humans [1]. High

performance liquid chromatography (HPLC), gas chromatography (GC) with MS detection, and a high-throughput sensor array were used for this purpose [2-4]. However, most assays suffer from some drawbacks such as a tedious sample pretreatment process, large sample volume required, organic solution needed for extraction, or high cost. Hence, it is urgent to develop simple, environmentally friendly, high-throughput methods for simultaneous detection of multiplex antibiotic

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residues in food. In recent years, biosensors that use aptamer as recognition

component called an aptasensor have attracted great attention for use in food safety [5,

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6]. An aptamer is a single-stranded DNA (ssDNA) or RNA fragment with special

base sequences [7]. Compared to other recognition components such as antibodies, it has many distinctive features, including higher affinity, ease of mass production by

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PCR, excellent specificity, and flexibility for modification [8, 9]. Moreover, aptamers

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can be easily designed to recognize small molecules, e.g., antibiotics. Many kinds of

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colorimetric, fluorescence, and electrochemical aptasensors with excellent sensitivity and selectivity for detecting antibiotics have been developed [10-12].

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However, most reported antibiotic aptasensors were used to detect a single target.

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If aptasensors were designed to detect multiple targets, novel aptamer probes that produce different signals could be designed. Moreover, the corresponding signals would need to be separated or easily distinguishable. These requirements increase the

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difficulty of synthesizing a probe and the complexity of device. Recently, microchip electrophoresis (MCE) has begun to play an important role in analysis of multiple

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analytes in biomedicine due to its merits of high-throughput, small size and the minimal sample volume required, as well as high efficiency contrasting with traditional aptasensor [13]. Lately, MCE has been introduced into a variety of

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applications, including DNA analysis，protein and small molecular separation with high efficiency and reproducibility [14, 15]. For example, Lu et al. reported the use of microchip capillary electrophoresis for rapid analysis of several anthracyclines with detection limits of 0.3 and 0.2 μg/mL for doxorubicin and daunorubicin, respectively [16]. García et al. used MCE for direct detection of antibiotics with limits of 5 and 350 μM for penicillin and ampicillin, respectively [17]. Therefore, MCE is a powerful

platform for multiple antibiotic detection. However, there were still some issues to be resolved. First, antibiotics that can be detected are limited to those with intrinsic luminescence or electroactivity or charged molecules for separation [18]. Second, specially designed MCE chips should be designed for different types of targets, increasing the cost and limiting the applicability of MCE. Third, some target need be derivatized before being detected by MCE, which increase the complexity of the

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experiment[19, 20]. Fourth, the sensitivity of commercial MCE has been limited because nanoliter sampling volumes are used, and the separation channel is limited to

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a few microns [21]. To solve this problem, our group recently developed two

universal platforms based on MCE for antibiotic detection using aptamer probes [22, 23]. In both assays, we used an aptamer functionalized probe that could quantifiably

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convert the amount of target to that of DNA fragments with different lengths. Thus, a

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commercial cross-type microfluidic chip can be used to separate and detect DNA

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fragments as a universal platform for antibiotic detection. However, both assays were able to detect only one target in a single separation channel, limiting the throughput of

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the array. Recently, many aptamer functionalized magnetic beads have been

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employed in multiplex antibiotic detection [24, 25]. On this basis, we hoped to develop a novel MCE array-based aptasensor using multiple aptamers with functionalized magnetic beads for multiplex detection of antibiotics and signal

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amplification to increase sensitivity.

Several signal amplification approaches have been successfully applied to the

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design of sensitive aptasensor, including polymerase chain reaction (PCR) [26], hybridization chain reaction (HCR) [27], rolling circle amplification (RCA) [28], and nicking enzyme assisted signal amplification (NEASA) [29]. Among these methods,

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PCR shows excellent amplification ability, and it is widely applied for nucleotide signal amplification at low cost. Thus, in the assay we developed, PCR was chosen for signal amplification. However, the ability to accurately quantify a template in conventional PCR is affected by a number of variables during PCR amplification or in the detection of PCR products. Here, an internal standard strand was co-amplified by PCR with the released assistant DNA (A-DNA). As for the variations occurred in

detection, two DNA markers mixed with samples were analyzed at the same time by MCE to overcome interference by the sample matrix. Finally, the ratio of the above PCR products was used for quantification. In summary, we developed a novel MCE array-based aptasensor using magnetic beads that modified with multiple aptamer probes for multiplex detection of

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antibiotics, using kanamycin (KANA) and chloramphenicol (CAP) as models and PCR for signal amplification. The entire detection procedure is illustrated in Scheme

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1. Composite probes consisting of two capture DNA (C-DNA) and assistant DNA (A-DNA) that labeled on magnetic gold nanoparticles (AuMPs) were employed. The C-DNA was a long ssDNA composed of an aptamer and random sequence. The A-DNA was partially hybridized with the C-DNA. When the targets (KANA and

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CAP) were introduced into the system, their respective A-DNA were replaced in the

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supernatant after magnetic separation because of the higher affinity of C-DNA for the

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target than for A-DNA. Then, the released A-DNA was co-amplified by PCR with the

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internal standard strand (I-DNA). Then, PCR products with different lengths (50 bp for the A-DNA of KANA, 72 bp for A-DNA of CAP, 86 bp for the I-DNA) were

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separated and detected by MCE using two markers to overcome interference by the sample matrix. Finally, the ratio of the PCR products from A-DNA and I-DNA were

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used for quantification. This platform as equipped with four chips that can work simultaneously, based on this approach, an MCE array for multiple target detection

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was achieved. The analytical method has the following advantages: (1) the detection throughput can be obviously increased because more targets can be simultaneously detected in one channel of MCE. (2) It can provide a universal platform to detect

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different kinds of antibiotics including non-fluorescent or uncharged analytes without any derivatization. (3) The signal can be amplified by PCR in order to obviously increasing the sensitivity of MCE. Preferred position for Scheme.1 2. Experimental 2.1 Materials and reagents

The oligonucleotide sequences used in this strategy are shown in Table S1 and were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Fe3O4 nanospheres modified with amino (634 nm, 20 mg/mL) were purchased from Enriching Biotechnology, Ltd. (Shanghai, China). Kanamycin (KANA), chloramphenicol (CAP), streptomycin (STR), tetracycline (TET), erythromycin (ERY), and thiamphenicol (TAP) were purchased

magnetic

beads

was

phosphate-buffered

saline

(PBS)

(pH

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from Sigma-Aldrich Co., Ltd. (Milan, Italy). The blocking buffer solution used for the 7.4,

0.1

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KH2PO4-K2HPO4, 0.1 M KCl) containing 3% (w/v) bovine serum albumin (BSA). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was purchased from

Sigma-Aldrich Co., Ltd. (Milan, Italy). DNA-500 kits, containing DNA-500 marker

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(storage at −20°C) and DNA-500 separation buffer (storage at 2°C to 8°C) were

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obtained from Shimadzu Co., Ltd. (Kyoto, Japan). TE buffer solution (pH = 8.0,

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storage at 2°C to 8°C), SYBR Gold, SYBR GREEN and a 25-bp DNA ladder were purchased from Invitrogen Co., Ltd. (Shanghai, China). All other reagents were

2.2. Apparatus

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used throughout the study.

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analytical grade and used without further purification. Double-distilled water was

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Scanning electron micrographs (SEM) were obtained using a S3400N scanning electron microscope (Hitachi Co., Ltd., Japan). A transmission electron microscopic

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(TEM) image was obtained using a H600 transmission electron microscope (Hitachi, Japan). Dynamic Light Scattering (DLS) was performed using a Zetasizer Nano ZS90 (Malvin Instruments, Ltd., Malvin, UK). UV-vis spectra were recorded with a

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UV-1800 spectrophotometer from Shimazu Co., Ltd. (Kyoto, Japan). PCR was performed on a PCR instrument from LongGene Scientific Instruments Co., Ltd. (Hangzhou, China). Real-time PCR was performed in a Lightcycler 2.0 (Roche, USA). DNA products were separated and detected using a MultiNA 202 System purchased from Shimadzu Co., Ltd. (Kyoto, Japan). It was equipped with a blue LED light with an excitation wavelength of 470 nm and a fluorescence detector that could receive

luminous light at a wavelength of 525 nm. The voltage parameters used for MCE sampling and separation are shown in Table S3.

2.3 Treatment of real samples Different brands of milk and different kinds of fish were purchased from a local supermarket in Ningbo, China. The milk extraction was carried out by enzyme-linked

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immunosorbent assay (ELISA). First, 20 mL milk sample was measured and

centrifuged for 10 min at 2500 × g and 4°C to remove fat form the milk. Subsequently,

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2 mL sodium nitroprusside and sulfate zinc solution were added, and the mixture was

mixed thoroughly by shaking and centrifuged again for 10 min at 2500 × g and 15°C. Finally, 1 mL supernatant was used for detection by the assay. The extraction from

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fish was carried out according to a previous report [30]. First, 2.5 g fish sample, 2.5 g

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anhydrous sodium sulfate, and 5 mL ethyl acetate were mixed with a meat grinder for

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1 min. Then, the mixture was centrifuged for 5 min at 2000 × g, the supernatant was extracted by ethyl acetate (2 × 10 mL), and the extract was dried in a rotary

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evaporator at 50°C under vacuum. Second, 1 mL n-hexane and 1 mL acetonitrile were

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added for the extraction, and the n-hexane phase was discarded. Finally, the acetonitrile phase was evaporated under a stream of dry nitrogen at 50°C, and the

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dried residue was dissolved in 1 mL 0.1 M PBS buffer (pH 7.4) for further use.

3. Results and discussion

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3.1. Feasibility of the developed method Twenty microliters of KANA and CAP (1 nM) were introduced into the solution

with 200 μL (~9.0 mg/mL) magnetic composite probes (AuMPs@C-DNA@A-DNA).

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The specific synthesis steps were described in supplementary materials. It is illustrated in the Fig. 1A curve a, with two strong signals of PCR products produced by A-DNA, indicating that the A-DNA were replaced by targets and that the amplification efficiency of PCR was very good. In curve b, there was almost no signal from the supernatant without PCR amplification, which might be because of the small amount of replaced A-DNA after introduction of the targets; this did not reach the

MCE quantification limit. A blank control (curve c, no target, but with PCR) was also carried out. The signals were much weaker than those observed in curve a. We inferred that this background signal might be due to the high amplification efficiency of PCR. Fortunately, the signal intensity was almost same in each control sample after several repeated experiments; thus, this amount could be deducted and did not interfere with detection. The detailed process of separation and detection of MCE

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were described in our previous report [22].

Accurate quantification in conventional PCR is affected by a number of variables

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during PCR or in the detection of PCR products. Here, an internal standard strand was co-amplified with the corresponding released A-DNA to revise the amplification efficiency between different reaction tubes. Thus, if the standard and target are

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amplified with the same efficiency, the ratio (IA-DNA/II-DNA) of products following

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PCR will reflect the amounts present, enabling absolute quantification. As illustrated

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in Fig. 1B, the product of the internal standard strand with a constant initial amount (0.01 nM) was almost stable after 20 cycles, and when different concentrations (5 nM,

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1 nM, 0.5 nM) of the target were added to the samples, the ratio between above

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products was proportional. As for variation occurring during detection procedure, two DNA markers mixed with samples were analyzed by MCE at the same time to reduce interference by the sample matrix. All of these methods were fully validated and

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found feasible. Therefore, with the help of the MCE platform and PCR, we

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successfully detected two antibiotics. Preferred position for Fig.1

3.2 Optimization and characterization of the AuMPs@C-DNA To verify that AuNPs were successfully conjugated with Fe3O4 to form AuMPs,

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the composites was characterized by SEM (Fig. 1A, B) and TEM (Fig. 1C). Moreover, UV-vis spectrophotometry was employed to demonstrate successful immobilization of C-DNA on AuMPs to form AuMPs@C-DNA (Fig. 1D). As shown in Fig. 1A, the surfaces of spherical Fe3O4NPs with an average diameter of 634 nm were smooth. The morphology of AuMPs is shown in Fig. 1B. A large number of AuNPs (Fig. 1C) were successfully attached to the surfaces of the Fe3O4NPs, which provided many

active sites for aptamers because of gold-sulfur chemistry. The nanoparticles were further confirmed by UV-vis absorption spectrum as shown in Fig. 1D. The UV spectrum of Fe3O4NPs (curve a) showed almost no absorption from 200 nm to 700 nm. After AuNPs with an average size of 35 nm were modified on the surfaces of Fe3O4NPs, an obvious characteristic peak of Au was observed at 550 nm (curve c), which showed a slight red shift compared to that with bare AuNPs (curve b). After

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reacting with C-DNA, curve d showed an obvious absorption peak at 260 nm, which

was attributable to the characteristic absorption peak of DNA compared to that of

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curve c. Those results indicated that the AuMPs@C-DNA complex was successfully synthesized. The DLS images and elemental analysis of particles are shown in Fig. S2.

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The amounts of added AuNPs were optimized in Fig. S3. After mixing volumes

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of AuNPs (2.9 × 10-8 mol/L, from 0 mL to 180 mL) with Fe3O4 (2 mL, 20 mg/mL)

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and shocking for 20 minutes, the color of the supernatant was almost transparent after magnetic separation (Fig.S3A). This meant that the AuNPs were totally adhered to

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Fe3O4. With the continuous addition of volumes from 180 mL to 220 mL, the color of

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the supernatant gradually turned to wine red (Fig.S3B, C), indicating that the absorption of Fe3O4 to AuNPs has reached saturation. The supernatant after magnetic separation was further examined by UV-vis absorption spectrum, and the absorption

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peak at 550 nm is shown in Fig. S3D. This indicated the optimum volume of AuNPs

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was about 180 mL.

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3.3 Optimization of assay conditions In order to increase the efficiency of the developed method, reaction time and

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temperature played a vital role. As shown in Fig. S4, according to the amount of the hybridization chain (dsDNA), consisting of C-DNA and A-DNA, in the system, the optimum hybridization reaction time was 15 min and the optimal temperature was 25°C (Fig. S4A, B). It was worth noting that above 25°C, the amount of dsDNA decreased, which may be due to the amount of base pairing between C-DNA and A-DNA was limited. After the targets were introduced, the amount of dsDNA

decreased because the A-DNA was replaced by the targets with higher affinity to C-DNA. Therefore, the best replacement time was 30 min, and 20°C was the most optimal temperature (Fig.S4C, D). Another key parameter that affected the assay performance was the complementary bases of A-DNA and C-DNA. The different A-DNA are listed in Table S1. When there were few complementary bases to C-DNA, A-DNA would not hybridize with

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C-DNA stably to form dsDNA; if there were too many complementary bases, A-DNA could not be easily replaced from the hybrid duplex by the target. Therefore, five

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A-DNA with 8 to 16 bases complementary to those in the C-DNA were detected. Fig. S5(A) showed that both A-DNA 1 and 2 for two targets provided good signals from

the dsDNA. Hence, they were used for the reaction with targets. The results are

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shown in Fig.S5(B). It was found that their amplification efficiencies were

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comparable. In consideration of the stability of the dsDNA, A-DNA 1 of KANA and

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CAP were chosen for the next experiment. Thus, the optimized number of complementary bases between A-DNA and C-DNA was 16.

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To determine the optimum PCR cycle, several experiments were carried out.

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A-DNA (0.1 nM, 0.5 nM, 1nM) was co-amplified with the internal standard strand (0.01 nM), and the ratio of PCR products was calculated as shown in Fig. S6. The ratio showed good correlation (R2 = 0.9952) with the amount of A-DNA when 20

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cycles were performed. When the cycle number was 25 or 30, the R2 was less than 0.9. This might indicate that the PCR of A-DNA reached saturation. Therefore, 20 was the

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optimum cycle of PCR cycles.

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3.4. Analytical performance of the assay To evaluate the analytical performance of the proposed strategy, the PCR products

were measured in the presence of the target at various concentrations. Under the optimal experimental conditions, the ratio between the PCR products of A-DNA and I-DNA gradually increased with an increase in the target concentrations, and the calibration curve was constructed according to the above relationship. The linear curve fit a regression equation of Y =39.38 + 3.184 log CKANA, with a correlation

coefficient represented by R2 = 0.9929 for KANA. The liner range was 0.0025–10 nM. The equation for CAP was represented as Y = 35.18 + 2.938 log CCAP, with a correlation coefficient of R2 = 0.9947 and a liner range of 0.006–10 nM. The method demonstrated optimal sensitivity with detection limits up to 0.0025 nM (KANA) and 0.006nM (CAP). Other determination assays for KANA and CAP detection are shown in Table 1. Compared with these, our strategy exhibited a wider linear range and

application in the detection of ultra-trace level of antibiotics.

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lower detection limit. Therefore, this biosensor showed great promise for practical

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In addition, real time PCR was also performed to compare with the results

obtained from the developed method (Fig.S7). The specific process was described in supplementary materials, and the results comparison was shown in Table.S4. It was

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found that the results were basically consistent at antibiotics’ concentration higher

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than 0.1 nM, but still had some disparity in lower concentration (<0.1 nM). It was

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inferred that this method may only achieve semi-quantitative detection in certain range of testing (<0.1 nM). Nevertheless, the quantitative detection range (＞0.1 nM)

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of this method was suitable for detecting antibiotics in food sample based on some stipulations. For example, the United States Food and Drug administration has

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decided the “minimum required performance limit” (MRPL) of CAP as 0.3 μg/kg for the detection of its residues in food products. And according to the government

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standard of China (GB/T 22969-2008), the MRPL of KANA in milk was 10.0 μg/kg. These values were both higher than 0.1 nM, which meant that this analytical method

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can be used for antibiotics detection in food sample.

Preferred position for Fig.3 Preferred position for Table.1

3.5. Selectivity of detection We also employed four other antibiotics, including streptomycin (STR), thiamphenicol (TAP), tetracycline (TET), and erythromycin (ERY), as interference in a selectivity experiment. Among them, STR and KANA are aminoglycoside

antibiotics, and TAP and CAP have similar chemical formulas. The remaining disturbances (TET and ERY) belonged to different antibiotic classes. Each was added to the incubation system at an identical concentration (1 nM), and the mixtures consisting of the above antibiotics and targets were tested. As shown in Fig. 4, the solutions contained KANA (0.5 nM) and CAP (0.5 nM) showed the highest signals. This indicated that the aptasensor could detect KANA and CAP with high specificity.

supplementary materials.

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Preferred position for Fig.4

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And the precision, accuracy, and high-throughput features of detection were shown in

3.6. Application to real samples

To confirm the practical utility of the developed method, we challenged the

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biosensor with several milk and fish samples. In Table 2, the acceptable recovery is

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shown to range from 94.40% (87.40%) to 105.6% (103.2%) for KANA and

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from85.4% (91.13%) to 106.8% (104.3%) for CAP in milk (fish). These results

samples with good accuracy.

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indicated that the sensing system can be applied to antibiotic detection in food

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4. Conclusion

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Preferred position for Table.2

In summary, a universal MCE array-based aptasensor for multiplex antibiotics

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detection was created. To develop this assay, novel multiple aptamer-functionalized magnetic beads were designed, and PCR was used for signal amplification. One internal standard strand and two markers were used to overcome variation that occur

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during PCR or in the detection of PCR products. The results compared with q-PCR showed that only semi-quantitative detection was achieved in certain range of testing (<0.1 nM). And when antibiotics’ concentration was higher than 0.1 nM, the results were basically consistent. For this problem, we will try to improve the technique in the next study. The average detection time for one antibiotic in one sample was about 1 min, making this MCE system high throughput. Furthermore, the assay showed

excellent resistance to interference by the same and different classes of antibiotics and proved to be an efficient platform for multiplex antibiotic detection for food safety. This assay can also be employed to detect other targets by changing the respective aptamer. Thus, it can serve as a useful and universal platform to screen various targets in food.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China

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(No.51403110), the Natural Science Foundation of Zhejiang and Ningbo

(LY17C200007，2017C33004), the plan of scientific and technological innovation activities for college students in Zhejiang province (2016A610084), and K.C.Wong

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Magna Fund in Ningbo University.

References

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[1] R. Singh, L. Sripada, R. Singh, Side effects of antibiotics during bacterial infection: mitochondria,

M

the main target in host cell, Mitochondrion, 16(2014) 50-4.

[2] K. Reddersen, T. Heberer, Multi‐ compound methods for the detection of pharmaceutical residues in various waters applying solid phase extraction (SPE) and gas chromatography with mass

ED

spectrometric (GC‐ MS) detection, Journal of Separation Science, 26(2003) 1443-50. [3] P. Olszowy, M. Szultka, J. Nowaczyk, B. Buszewski, A new way of solid-phase microextraction fibers preparation for selected antibiotic drug determination by HPLC–MS, Journal of Chromatography

PT

B, 879(2011) 2542-8.

[4] H. Liu, N. Gan, Y. Chen, T. Li, Y. Cao, Three dimensional M× N type aptamer-functionalized solid-phase micro extraction fibers array for selectively sorptive extraction of multiple antibiotic

CC E

residues in milk, RSC Advances, 7(2017) 6800-8. [5] R. Sharma, K. Ragavan, M. Thakur, K. Raghavarao, Recent advances in nanoparticle based aptasensors for food contaminants, Biosensors and Bioelectronics, 74(2015) 612-27. [6] L. Zhou, J. Wang, D. Li, Y. Li, An electrochemical aptasensor based on gold nanoparticles dotted graphene modified glassy carbon electrode for label-free detection of bisphenol A in milk samples,

A

Food chemistry, 162(2014) 34-40. [7] C. Marimuthu, T.-H. Tang, J. Tominaga, S.-C. Tan, S.C. Gopinath, Single-stranded DNA (ssDNA) production in DNA aptamer generation, Analyst, 137(2012) 1307-15. [8] A.D. Keefe, S. Pai, A. Ellington, Aptamers as therapeutics, Nature reviews Drug discovery, 9(2010) 537. [9] E.J. Cho, J.-W. Lee, A.D. Ellington, Applications of aptamers as sensors, Annual Review of Analytical Chemistry, 2(2009) 241-64. [10] M. Ramezani, N.M. Danesh, P. Lavaee, K. Abnous, S.M. Taghdisi, A novel colorimetric

triple-helix molecular switch aptasensor for ultrasensitive detection of tetracycline, Biosens Bioelectron 70(2015) 181-7. [11] H. Li, D.-e. Sun, Y. Liu, Z. Liu, An ultrasensitive homogeneous aptasensor for kanamycin based on upconversion fluorescence resonance energy transfer, Biosens Bioelectron 55(2014) 149-56. [12] Z. Yan, N. Gan, T. Li, Y. Cao, Y. Chen, A sensitive electrochemical aptasensor for multiplex antibiotics detection based on high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted cascade target recycling, Biosens Bioelectron 78(2016) 51-7. [13] L. Baraban, D. Makarov, R. Streubel, I. Monch, D. Grimm, S. Sanchez, et al., Catalytic Janus motors on microfluidic chip: deterministic motion for targeted cargo delivery, ACS nano, 6(2012)

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3383-9.

[14] S.D. Noblitt, L.C. Staicu, C.J. Ackerson, C.S. Henry, Sensitive, selective analysis of selenium oxoanions using microchip electrophoresis with contact conductivity detection, Anal Chem 86(2014)

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8425-32.

[15] D. Wu, J. Qin, B. Lin, Electrophoretic separations on microfluidic chips, Journal of Chromatography A, 1184(2008) 542-59.

[16] H. Lu, G. Yuan, Q. He, H. Chen, Rapid analysis of anthracycline antibiotics doxorubicin and daunorubicin by microchip capillary electrophoresis, Microchemical Journal, 92(2009) 170-3.

U

[17] C.D. García, C.S. Henry, Direct determination of carbohydrates, amino acids, and antibiotics by microchip electrophoresis with pulsed amperometric detection, Analytical chemistry, 75(2003)

N

4778-83.

[18] H.-L. Meng, G.-H. Chen, X. Guo, P. Chen, Q.-H. Cai, Y.-F. Tian, Determination of five quinolone

A

antibiotic residues in foods by micellar electrokinetic capillary chromatography with quantum dot

M

indirect laser-induced fluorescence, Anal BioanalChem 406(2014) 3201-8. [19] B.H. Huynh, B.A. Fogarty, P. Nandi, S.M. Lunte, A microchip electrophoresis device with on-line microdialysis

sampling

and

on-chip for

amino

acid

derivatization and

peptide

by analysis,

naphthalene Journal

of

ED

2,3-dicarboxaldehyde/2-mercaptoethanol

sample

pharmaceutical and biomedical analysis, 42(2006) 529-34. [20] P. Nandi, D.E. Scott, D. Desai, S.M. Lunte, Development and optimization of an integrated PDMS

PT

based-microdialysis microchip electrophoresis device with on-chip derivatization for continuous monitoring of primary amines, Electrophoresis, 34(2013) 895-902. [21] Y. Qin, L. Zhang, S. Li, J. Zhao, Y. Huang, S. Zhao, et al., A microchip electrophoresis-based

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fluorescence signal amplification strategy for highly sensitive detection of biomolecules, Chem Commun 53(2017) 455-8. [22] L. Zhou, N. Gan, Y. Zhou, T. Li, Y. Cao, Y. Chen, A label-free and universal platform for antibiotics detection based on microchip electrophoresis using aptamer probes, Talanta, 167(2017) 544-9.

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[23] Y. Wang, N. Gan, Y. Zhou, T. Li, F. Hu, Y. Cao, et al., Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification, Biosensors and Bioelectronics, 97(2017) 100-6. [24] M. Chen, N. Gan, Y. Zhou, T. Li, Q. Xu, Y. Cao, et al., A novel aptamer-metal ions-nanoscale MOF based electrochemical biocodes for multiple antibiotics detection and signal amplification, Sens Actuators B: Chem, 242(2017) 1201-9. [25] C. Liu, C. Lu, Z. Tang, X. Chen, G. Wang, F. Sun, Aptamer-functionalized magnetic nanoparticles

for simultaneous fluorometric determination of oxytetracycline and kanamycin, Microchimica Acta, 182(2015) 2567-75. [26] E. Fiore, E. Dausse, H. Dubouchaud, E. Peyrin, C. Ravelet, Ultrafast capillary electrophoresis isolation of DNA aptamer for the PCR amplification-based small analyte sensing, Frontiers in chemistry, 3(2015). [27] F. Xuan, I.-M. Hsing, Triggering hairpin-free chain-branching growth of fluorescent DNA dendrimers for nonlinear hybridization chain reaction, Journal of the American Chemical Society, 136(2014) 9810-3. [28] M.M. Ali, F. Li, Z. Zhang, K. Zhang, D.-K. Kang, J.A. Ankrum, et al., Rolling circle

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amplification: a versatile tool for chemical biology, materials science and medicine, Chemical Society Reviews, 43(2014) 3324-41.

[29] Y. Huang, J. Chen, S. Zhao, M. Shi, Z.-F. Chen, H. Liang, Label-free colorimetric aptasensor

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based on nicking enzyme assisted signal amplification and DNAzyme amplification for highly sensitive detection of protein, Analytical chemistry, 85(2013) 4423-30.

[30] M. Takino, S. Daishima, T. Nakahara, Determination of chloramphenicol residues in fish meats by liquid chromatography–atmospheric pressure photoionization mass spectrometry, Journal of Chromatography A, 1011(2003) 67-75.

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[31] N. Zhou, J. Zhang, Y. Tian, Aptamer-based spectrophotometric detection of kanamycin in milk, Analytical Methods, 6(2014) 1569-74.

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[32] C. Wang, D. Chen, Q. Wang, R. Tan, Kanamycin detection based on the catalytic ability enhancement of gold nanoparticles, Biosensors and Bioelectronics, 91(2017) 262-7.

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[33] S. Pilehvar, T. Dierckx, R. Blust, T. Breugelmans, K. De Wael, An electrochemical impedimetric Sensors, 14(2014) 12059-69.

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aptasensing platform for sensitive and selective detection of small molecules such as chloramphenicol, [34] Y. Liu, K. Yan, O.K. Okoth, J. Zhang, A label-free photoelectrochemical aptasensor based on

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nitrogen-doped graphene quantum dots for chloramphenicol determination, Biosensors and

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Bioelectronics, 74(2015) 1016-21.

Biographies Lingying Zhou entered the MS program in 2015, majoring in food Science and biosensor. Ning Gan earned his Bachelor and PhD degrees in analytical Chemistry from Nanjing

China. His research interests are in the areas of biochips and biosensors.

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University, China. He is a professor at chemistry department in Ningbo University,

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Futao Hu earned his Bachelor and Master degrees in Analytical Chemistry from Zhejiang University, China. His research interests are in the areas of food safety

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inspection technology.

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Yuting Cao received his MS degree from Tongji University. He is engaged in

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bioanalysis.

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Tianhua Li earned his MS degree in Analytical Chemistry from Liaocheng University,

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Shandong, China. He is engaged in the analysis of biomaterials and foods. Dazhen Wu earned his Master and PhD degrees in Analytical Chemistry from

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University of Science and Technology of China，China. Her research interests are in

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the areas of food safety inspection technology.

Caption Scheme 1. Overview of the aptasensor developed for simultaneous detection of antibiotic. Fig. 1. The feasibility of the developed method. (A) Curve a, a sample including targets (1 nM) with PCR; curve b, a sample including targets (1 nM) without PCR; curve c, a sample without targets, but amplified by PCR. (B) The migration index of the PCR product, including an internal

curve c: 0.5 nM).

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standard strand (0.01 nM) and target with different concentration (curve a: 5 nM, curve b: 1nM,

Fig. 2 SEMs of (A) Fe3O4NPs and (B) Fe3O4NPs@Au (AuMPs), TEM of (C) AuNPs and (D) the

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UV-vis spectra of (a) Fe3O4NPs, (b) AuNPs, (c) AuMPs, and (d) AuMPs@C-DNA.

Fig. 3. Linear ratio changes with targets concentrations. (A) Calibration plot in the presence of different concentrations (10 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.0025 nM) of KANA; (B)

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Calibration plot in the presence of different concentrations (10 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01

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nM, 0.006 nM) of CAP.

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Fig. 4. Selectivity experiments for targets (0.5 nM) against other antibiotics, including STR, TET, ERY, and TAP at final concentrations of 1 nM. (A) Organic compounds of the targets and other

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Scheme.1

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antibiotics. (B) Signals from the samples with different antibiotics.

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Fig.1

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Fig.2

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Fig.3

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Fig.4

Table 1. The assay compared with other methods for the detection of KANA and CAP residues. Detection limit (pM) KANA CAP 1000 —

—

0.1–20

Reference [31]

100

—

[32] [33] [34]

— —

1.76–127 10–250

— —

1760 3100

0.0025–1 0

0.006–10

0.0025

0.006

This method

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Spectrophotometric detection Colorimetric detection based on gold nanoparticle Electrochemical aptasensor Photoelectrochemical aptasensor This work

Linear range (nM) KANA CAP 1–500 —

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Method

Added (nM)

Detection (nM)

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Blank (nM)

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Table2. Detection of KANA and CAP in real sample (n = 3)

CAP

KANA

Milk 1

0.068 0.063

0.5

Milk 2

0.056 0.057

1.5

Milk 3

0.049 0.046

Fish 1

0.053 0.056

Fish 2 Fish 3

KANA

CAP

Recovery (%)

KANA

CAP

0.54 ± 0.042

0.49± 0.027

94.40

85.4

1.5

1.64 ± 0.032

1.66 ± 0.043

105.6

106.8

5.0

5.0

4.93 ± 0.045

5.23 ± 0.034

97.62

103.7

0.5

0.5

0.49 ± 0.047

0.53± 0.025

87.40

94.80

0.072 0.063

1.5

1.5

1.62 ± 0.036

1.43 ± 0.054

103.2

91.13

0.083 0.074

5.0

5.0

5.14 ± 0.038

5.29 ± 0.038

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104.3

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CAP

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KANA

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Sample