Palindromic Fragment-Mediated Single-Chain Amplification: An

May 22, 2019 - The LOD value was approximately 22 fM at a signal-to-noise ratio of 3. Distinctly, here the as-proposed strategy for Kana assay could d...
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Article Cite This: Anal. Chem. 2019, 91, 7835−7841

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Palindromic Fragment-Mediated Single-Chain Amplification: An Innovative Mode for Photoelectrochemical Bioassay Ruijin Zeng, Lijia Zhang, Zhongbin Luo, and Dianping Tang* Key Laboratory of Analytical Science for Food Safety and Biology (MOE & Fujian Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, People’s Republic of China

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S Supporting Information *

ABSTRACT: This work reports a strategy for glutathione-loaded liposome-encoded magnetic beads initiated by palindromic fragment-mediated single-chain amplification (PFMSCA) for highprecision quantification of a low-abundance aminoglycoside antibiotic (kanamycin; Kana) by using In2O3−ZnIn2S4 (IO-ZIS) as a photoactive matrix. In this strategy, a Kana-recognition region, primer-like palindromic fragment, and polymerization/nicking template are reasonably integrated into one oligonucleotide (hairpin HP1) for target recognition, magnetic separation, and target amplification. Upon target Kana introduction, the Kana-aptamer region in HP1 specifically recognizes the Kana and triggers the palindromic tails intramolecular self-hybridization, amplifying a large number of short fragments in the presence of Klenow fragment polymerase and Nt.BbvCI. The as-generated nick fragments act as a linker to introduce the free hairpin HP2-functionalized glutathione-loaded liposomes (HP2-GLL) onto the surface of the hairpin HP3-modified magnetic beads (HP3-MB), constructing liposome-encoded magnetic beads (HP3-MBnick-HP2-GLL). Following magnetic separation, the detached glutathione-loaded liposomes (GLL) are lysed by treatment with 1% Triton X-100 to release the glutathione within it, which were then detected as an amplified photocurrent at the IO-ZISbased photoelectrode. Importantly, this method can be readily carried out by using one oligonucleotide to achieve an exponential amplification effect and open new opportunities for advanced development of robust biodetection systems.

I

simplicity and powerful signal amplification while preserving requisite benefits in sensitivity and flexibility. Palindromic repeats sequence is a short inverted duplication nucleotide fragment located in gene untranslated operon and specific protein binding region.10 A prominent and fascinating feature of palindromic repeats sequence is the complementary fragment being a palindromic sequence of each other. Such fascinating properties enable it to act as a primer in the field of nucleic acid molecular sensing. For instance, Li et al. devised a newly palindromic fragment-incorporated molecular beaconbased fluorescence aptasensor for detection of tumor suppressor gene p53.11 Shen et al. established a sensing system for the convenient and sensitive colorimetric bioassay detection of cancer genes based on a multifunctional molecular beacon with a palindromic tail.12 However, the fascinating feature of this palindromic self-hybridization urgently needs to be extended for other non-nucleic acid target (NNAT) detection. Actually, analysis of NNAT can be converted to detection of an amplifiable DNA fragment, enabling sensitive high-capacity NNAT measurements using different DNA amplification technologies. Recently, our laboratory constructed a desirable sensing system for the ultrasensitive and simple detection of antibiotic by the method of palindromic

n 1992, Walker and co-workers proposed the concept of strand-displacement amplification (SDA), a unique combination of DNA polymerase and restriction endonuclease DNA amplification technique.1 Since then diverse schemes involving SDA approaches have been reported in the field of basic research as well as diagnostic/monitoring applications due to its simplicity, versatility, robustness, and independence from thermal cyclers.2−4 Recently, our research group ingeniously constructed an ultrasensitive electrochemical/piezoelectric aptasensor for low-dose biological targets detection combining target-induced-SDA with other isothermal amplification strategies (hybridization chain reaction, Exo III-assist target recycling, and catalytic hairpin assembly).5,6 Unfortunately, we clearly noticed that the SDA-based approach requires sophisticated primer design and cumbersome procedures and is usually accompanied by a risk of nonspecific background amplification (NSBA). Specifically, one or more primers involved in the system for target recognition and/or target amplification inevitably increase the likelihood of primerdimer-related NSBA resulting from unintended Watson−Crick interactions,7,8 namely, employment of primer and/or other template requires very tight control of their extension, cleavage, and strand displacement process in order to attain reproducible signals readout.9 These drawbacks have always limited their practical application in basic research in terms of repeatability, sensitivity, and universality. To tackle such weakness associated with NSBA and sophisticated probe design, there is still the requirement to explore design © 2019 American Chemical Society

Received: March 28, 2019 Accepted: May 20, 2019 Published: May 22, 2019 7835

DOI: 10.1021/acs.analchem.9b01557 Anal. Chem. 2019, 91, 7835−7841

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Scheme 1. Schematic Illustration of Glutathione-Encapsulated Liposome-Encoded Magnetic Beads Initiated by Palindromic Fragment-Mediated Single-Chain Amplification for Quantification of Kanamycin (Kana) Using In2O3−ZnIn2S4 (IO-ZIS) as Photoactive Materiala

a

Kana-apt: base sequence corresponding to Kanamycin-aptamer. HP1: hairpin DNA1. HP2-GLL: hairpin DNA2-functionalized glutathione-loaded liposomes. HP3-MB: hairpin DNA3-functionalized magnetic beads. GSH: glutathione. Nick: cleavage DNA.

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molecular beacons-mediated Z-scheme photoelectrochemical (PEC) biodetection.13 Although we introduced the palindromic sequence into the hairpin molecule beacon to replace the primer in the previous work, we also designed two additional nucleotides for target recognition. As a continuation and improvement of previous work, we further integrate the function of target recognition into the palindrome beacon and only one oligonucleotide is involved in the entire SDA process. Kanamycin (Kana; a powerful aminoglycoside-type over-thecounter antibiotic drug) fights bacterial infections against Gram-negative pathogens and plays important roles in the pharmaceutical industry, human health, and food safety.5 Accumulating evidence has revealed that overuse/misuse of Kana in poultry farming areas is closely associated with the development of drug-resistant bacterial strains and other adverse reactions.6,13 Accordingly, accurate quantification, instant analysis, and cost-efficient detection of antibiotic residue is of critical significance in pharmacological and biochemical research. Toward this goal, herein we design a simple and high-performance PEC sensing platform for the detection of Kana (Scheme 1). Palindromic fragment-mediated single-chain amplification (PFMSCA)-based target recycling is used for signal amplification on the In2O3−ZnIn2S4 (IO-ZIS) photoactive matrix. In the sensing system a trace amount of target Kana can be converted to numerous glutathione (GSH) molecules, leading to a remarkable amplification via PFMSCA and the liposome-encoded enzyme catalytic.14 In this research, we take full advantage of the characteristics that palindromic endows a new concept of development of a powerful PEC platform for screening of low-concentration biomolecules.

EXPERIMENTAL SECTION

Preparation of Glutathione-Loaded Liposome. Glutathione-loaded liposome (GLL) was synthesized on the basis of the previous literature with minor modifications.14 In a typical procedure, the DSPC, cholesterol, and DSPE-PEG-COOH (molar ratio 7:1:2) were dissolved into a mixture containing chloroform (5.0 mL). Then the mixture was evaporated at 45 °C in a rotary evaporator until a gel-like suspension was obtained. Following that the glutathione solution (10 mL, 1 mM) was injected into the above suspension. Afterward, GLL was formed after sonication for 4 min. Last, the GLL was extruded through a 0.22 μm polycarbonate filter to produce a homogeneous suspension with uniform size. The suspension of the prepared liposomes was stored in a refrigerator before use. During synthesis, the antioxidant tripeptide glutathione molecules were encapsulated into the liposome by a reversed-phase evaporation method similar to our previous reports.15−17 Preparation of HP3-Modified Magnetic Beads and HP2-Functionalized GLL. The carboxyl-functionalized GLL was activated using EDC (25 μL, 0. One mM) and NHS (25 μL, 25 μM) for 4 h. Then HP2 (20 μL, 50 nM) was added and incubated for 8 h in ambient temperature with gentle shaking. Finally, the as-obtained HP2-GLL was purified by centrifuged for 10 min at 10 000g to remove the unreacted HP2. Hairpin HP3 is bound to the magnetic beads by a cross-linking reaction between the streptavidin-functionalized magnetic beads (MB) and biotinylated cDNA. MB (50 μL, 1 mg mL−1) were first rinsed and then resuspended in PBS buffer (pH 7.4, 500 μL). Subsequently, hairpin HP3 (100 nM) was added and 7836

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Figure 1. (A) TEM of IO-ZIS. (B) SEM images of ZIS (inset, magnification image). (C) SEM image of IO-ZIS (inset, magnification image). (D) HRTEM image of IO-ZIS (inset, SAED pattern). (E) HAADF-STEM image and element mapping for In (green), O (yellow), Zn (red), and S (blue) of IO-ZIS. XPS survey spectra of (F) IO-ZIS (inset, high-resolution XPS spectra of Zn 2p and In 3d), (G) O 1s, and (H) S 2p. (I) N2 gas adsorption−desorption isotherms of IO-ZIS (inset, pore-size distribution).

thermostatic metal bath for 45 min. Then the unreacted HP2-GLL and HP3-MB were removed with magnetic separation followed by washing the resultant HP3-MB-nickHP2-GLL three times. Next, 10 μL of 1% Triton X-100 was added into the resultant HP3-MB-nick-HP2-GLL and maintained for 13 min to lyse the captured liposomes for liberating the encapsulated GSH. Following that the resulting product containing the as-released GSH from the GLL was transferred into the homemade detection cell (in 5.0 mL 10 mM PBS, pH = 7.4) for measurement. Fabrication of PEC Platform and Measurement. IOZIS-coated fluorine-doped tin oxide (IO-ZIS/FTO) electrode was prepared as follows. First, brand-new FTO glass was washed in ethanol and deionized water and dried at ambient temperature. Thereafter, the cleaned FTO was attached with a waterproof transparent tape of 2.5 mm in radius processed by a puncher. Following that the above-prepared IO-ZIS aqueous suspension (30 μL, 10 mg mL−1) was thrown on the surface of FTO and dried at ambient temperature. Then the photocurrent was measured in a homemade detection cell on an electrochemical workstation.

incubated for 2 h with gentle shaking. The resultant HP3-MB was then rinsed with PBS three times. Synthesis of In2O3−ZnIn2S4 Heterostructures. In2O3− ZnIn2S4 (IO-ZIS) was synthesized on the basis of the previous literature with minor revision.18 In detail, 120 mg of In(NO3)3· xH2O and 120 mg of 1,4-benzenedicarboxylic acid were dissolved in 80 mL of DMF under vigorous stirring. Then the above solution was placed in an oil bath at 120 °C for 0.5 h. The obtained white precipitate was annealed in air at 500 °C for 2 h (heating rate 5 °C min−1), and the IO with a light yellow color was finally obtained. Growth of ZIS on surfaces of IO was achieved by a typical low-temperature hydrothermal method. First, IO solution (15 mg, 20 mL, pH = 2.5) was added and stirred for 30 min, followed by addition of three ZIS precursors (88.4 mg of InCl3, 54.4 mg of ZnCl2, and 60 mg of thioacetamide). The obtained mixture was stirred and put into an oil bath at 80 °C for 2 h. After that the resulting products (i.e., IO-ZIS) were collected by centrifugation for 10 min at 10 000g, washed with ultrapure water thoroughly, and dried at 65 °C in a vacuum oven. Target-Triggered PFMSCA-Based Target Recycling. Prior to experiment, 5.0 μL of 100 μM HP1 was initially added into 95 μL of 10 mM PBS buffer (pH 7.4, 20 mM KCl, 200 mM NaCl, and 10 mM MgCl2). Thereafter, the resulting mixture was denatured for 3 min at 95 °C and finally cooled to ambient temperature. Next, 25 μL of PBS buffer containing the different concentration Kana, 2.5 μL of 10× NEBuffer, HP1 (5.0 nM), dNTPs (250 μM), KF polymerase (5.0 U), and Nt.BbvCI (5.0 U) were incubated at 37 °C in the thermostatic metal bath for 150 min. A 50 μL amount of mixture containing HP3-MB (0.2 mg mL−1) and HP2-GLL (0.2 mg mL−1) was first incubated with the nick DNA prepared by PFMSCA at 37 °C in a

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RESULTS AND DISCUSSION Characterization of ZIS, IO-ZIS, GLL, and HP2-GLL. Initially, the IO-ZIS nanosheets used in this study were easily prepared by the typical solvothermal method and lowtemperature hydrothermal reaction.18−20 A transmission electron microscopy (TEM) image shows that ZIS nanosheets are uniformly grown on the surfaces of IO with wellmaintained one-dimensional morphology (Figure 1A). Details regarding their top-view and cross-sectional morphological features were further studied through scanning electron microscopy (SEM). Figure 1B gives the morphologies of the 7837

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Figure 2. (A) TEM image of GLL on carbon grid. (B) DLS data of GLL. (C and D) 2D and 3D AFM images of GLL on mica plate. (E) UV−vis absorption spectra of (a) blank liposome and (b) HP2-GLL. (F) PAGE (8% gel) for different samples (lane M, 300 bp-marker; lanes a−c, Hp1− Hp3; lane d, HP1 + Kana + KF polymerase + Nt.BbvCI + dNTPs; lane e, lane d + HP2 + HP3). (G) Photocurrent responses of (a) ZIS/FTO, (b) IO-ZIS/FTO, (c) IO-ZIS/FTO incubated with blank liposome, and (d) IO-ZIS/FTO incubated with 0.5 nM GSH (inset, time-resolved transient PL decay of (a) ZIS and (b) IO-ZIS]. (H) Photocurrents of (a) IO-ZIS/FTO and (b) IO-ZIS/FTO without target and (c) with target (blue and orange columns indicate absence or presence of 1% Triton X-100). (Concentrations of Kana, KF polymerase, Nt.BbvCI, dNTPs, HP1, and HP2 were 5.0 nM, 5.0 U, 5.0 U, 250 μM, 5.0 nM, and 5.0 nM, respectively.)

signals at 161.9 (S 2p3/2) and 163.0 (S 2p1/2), confirming the existence of the S2− state (Figure 1H).26 Moreover, the Brunauer−Emmett−Teller (BET) surface area, porosity properties, and pore size distribution of IO-ZIS were measured by N2 gas adsorption−desorption isotherm. Obviously, the graph of IO-ZIS in Figure 1I is characteristic of a type IV isotherm (IUPAC classification), indicating that a mesoporous structure and slit-like pores existed within the material.27 The surface area of IO-ZIS is estimated to be 189.9834 m2 g−1, and the average pore size of IO-ZIS was approximately 3.3 nm (Figure 1I, inset), further implying the IO-ZIS has strong adsorption ability.28 Overall, the aforementioned results from TEM, SEM, HRTEM, EDS element mapping, XPS, and N2 gas adsorption−desorption isotherm undoubtedly confirm that IO-ZIS was successfully obtained via a facile hydrothermal approach and low-temperature hydrothermal reaction route in sequence. As described above, the nanometer-sized GLL was used as an enhancer to increase the photocurrent of IO-ZIS-based photoelectrode. To investigate the successful preparation of nanometer-sized GLL, negative-stain TEM (NS-TEM) was utilized to characterize its morphological profile. Figure 2A shows that the NS-TEM image of the as-prepared GLL on a carbon grid exhibited spherical or quasi-spherical shape with well-defined edges. The dynamic light scattering (DLS) measurement in Figure 2B shows that the range of GLL diameters spanned 80−250 nm, and the polydispersity index was 0.117, indicating that the GLL with a favorable size and uniform distribution could provide a vast space for GSH loading. Moreover, atomic force microscopy (AFM, 2D and 3D images) further validated the morphology profile of GLL. Images in Figure 2C and 2D illustrate that the GLL were well dispersed on the mica surface without any noticeable cracks and the shape with a size below 250 nm, which agrees quite well with the DLS and NS-TEM data. These results gave immediate evidence for successful preparation of the GLL. Furthermore, the free GLL and HP2-GLL were subjected to

as-synthesized ZIS by using ZnCl2, InCl3, and thioacetamide without the IO, which was mainly marigold-like with the size ranging from 0.5 to 2.2 μm. After hydrothermal treatment with IO, the IO-ZIS indicated that part of the rod-like IO gleamingly interspersed on marigold-like ZIS units to form micrometer flower-encased nanosheets (Figure 1C). In addition, the fringes with a lattice distance of 0.322 nm in the outer layer match well with the (102) plane of hexagonal ZIS, and the lattice spacing of about 0.292 nm could be assigned as the (222) crystal plane of cubic IO in the highresolution TEM (HRTEM) image (Figure 1D), providing good evidence of tight contact between IO and ZIS.21,22 To further testify the elemental distribution of the two substances, energy-dispersive X-ray (EDX) elemental mapping analysis was carried out in the IO-ZIS nanocomposites (Figure 1E). The corresponding element mappings over the scanning area certify the coexistence and uniform distribution of In (green), O (yellow), Zn (red), and S (blue) elements in the entire composite, reflecting formation of homogeneous lengthwise interfacial junctions between the IO shell and the ZIS layers. To obtain insight into the interfacial interaction and exact surface state between IO and ZIS in the composites, surfacesensitive X-ray photoelectron spectroscopy (XPS) was further carried out. As shown from the full XPS survey spectrum in Figure 1F, all element valences (S 2p; In 3d, 3p; O 1s; Zn 2p) related to IO and ZIS can be observed in IO-ZIS. The binding energies of Zn 2p3/2 and Zn 2p1/2 are 1021.85 and 1044.95 eV in the IO-ZIS (Figure 1F, top left inset), which can be attributed to the Zn2+ chemical state existing in the composites.23 Similarly, two individual peaks are situated at 445.4 and 453.0 eV (Figure 1F, top right inset), corresponding to In3+ 3d5/2 and In3+3d3/2 in IO-ZIS, respectively.24 Meanwhile, the O 1s peak of IO-ZIS was reasonably fitted with two component peaks located at 530.8 and 532.4 eV by using Gaussian fitting (Figure 1G), corresponding to the In− O−In and oxygen defects, respectively.25 Besides, the S 2p region of the IO-ZIS also presented the characteristic double 7838

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The photocurrent intensity progressively increased with continuously increasing Kana concentration over the range from 0.1 pM to 5.0 nM (Figure 3A), supporting the viability

UV−vis absorption spectroscopy (Figure 2E) to ensure attachment of HP2 to the surface of liposomes. No characteristic absorption peak was achieved at GLL alone (curve a), while a characteristic absorption peak at 260 nm (curve b) for HP2-GLL appeared, indicating the HP2 was successfully decorated on the GLL by typical carbodiimide reaction. Feasibility Investigation and Comparative Studies. For our design the change in the photocurrent value on the IO-ZIS-based photoelectrode was derived from the PMFSCAinduced GLL-encoded magnetic beads. Thus, the feasibility of the PMFSCA amplifier strategy for Kana assay was confirmed by 8% native-polyacrylamide gel electrophoresis (PAGE) (Figure 2F). Lanes a−c clearly gave a PAGE diagram of HP1−HP3, respectively. When the HP1 reacted with KF polymerase, Nt.BbvCI, and dNTPs, a series of different molecular weight gradient bands appeared thanks to intra/ intermolecular polymerization reaction (lane d). Upon further addition of the mixture of HP1 and HP2 into the lane d-based system, another bright spot for the newly formed stable HP2nick-HP3 complex could be achieved (lane e), which indicates that PMFSCA could take place as anticipated. According to the design of the scheme, the two next puzzling questions arise: (i) whether IO-ZIS nanosheets could have a strong photocurrent response to the GSH in the detection solution and (ii) whether Triton X-100 could cause the release of GSH from the GLL. To verify the concerns, a series of comparative studies was monitored at different conditions (Figure 2H and 2I). Chronoamperometric time−photocurrents spectra indicate that IO-ZIS exhibits a more greatly enhanced photocurrent than ZIS (curve b vs curve a in Figure 2H), revealing the promoted generation and transfer of photogenerated charge carrier in the IO-ZIS under visible light irradiation. In addition, the time-resolved transient PL decay spectra (Figure 2G, inset) revealed that the average emission lifetime of IO-ZIS (4.04 ns, curve a) is shorter than that of the corresponding ZIS (5.21 ns, curve b), providing solid evidence for the enhanced charge separation and transformation in IO-ZIS.29 Upon addition of GSH, a strong photocurrent significantly appeared in IO-ZIS (curve d vs curve b); however, no obvious change of the photocurrent occurred in the blank liposome (curve c vs curve b). On the basis of the above-mentioned finding and exploration, we might make the conclusion that only free GSH could amplify the photocurrent of IO-ZIS and blank liposome could not disturb the experimental phenomenon. Moreover, we further investigated the change of photocurrent before and after adding 1% Triton X-100 under different conditions. The photocurrent response in the absence of the target is similar to the IO-ZIS regardless of the presence or absence of 1% Triton X-100 (histograms a vs histograms b). As for the presence of the target Kana (5 nM), the photocurrent increased significantly after interaction with 1% Triton X-100 (histogram c). The increase in the photocurrent was attributed to the fact that as-released GSH from the GLL suppressed the electron− hole recombination of IO-ZIS. These results further revealed that our developed PFMSCA-PEC system could be utilized for detection of target Kana. Analytical Performance of PMFSCA-Based IO-ZIS PEC Biosensing Platform. By coupling PFMSCA-based signal amplification with a IO-ZIS-based PEC platform, a series of different concentration Kana standards was systematically tested under optimized reaction circumstances (Figure S1).

Figure 3. (A) Photocurrents of PFMSCA-based IO-ZIS-PEC bioanalysis platform toward target Kana. (B) Calibration plots between photocurrent (nA) and the logarithm of Kana concentration (pM). (C) Time-dependent photocurrents curve on IO-ZIS/FTO electrode under 15 on−off illumination for 300 s. (D) Antiinterference ability against 5.0 nM Kana, 50 nM cefixime (CFM), 50 nM vancomycin hydrochloride (VAN), 50 nM levofloxacin (LEV), 50 nM methicillin (MET), and the mixture containing the aforementioned analytes.

for Kana sensing. A preferable linear-dependence photocurrent density−time curve was acquired, and the corresponding equation was I (nA) = 72.17 × log C[Kana] + 419.84 (pM) with high reliability (R2 = 0.9902, n = 7) (Figure 3B). The LOD value was approximately 22 fM at a signal-to-noise ratio of 3. Distinctly, here the as-proposed strategy for Kana assay could display superior sensitivity compared with recently reported Kana schemes (Table 1), which mainly ascribed to smart Table 1. Comparison of Different Kana Scheme on Analytical Properties

a

methoda

linear range

LOD

ref

fluorescence PEC biodetection gas pressure assay electrochemical assay SERS assay PEC bioassay

2.0−5000 pM 1.0−5000 pM 0.2−50 pM 0.05−200 pM 1−100 nM 0.1−5000 pM

1.2 pM 0.78 pM 63 fM 36 fM 750 pM 22 fM

30 31 5 6 32 this work

SERS: surface-enhanced Raman scattering.

integration of the IO-ZIS photoactive matrix and PFMSCAmediated huge signal amplification approach endowing the platform with ultrasensitive photocurrent readout. The relative standard deviations (RSDs) were 7.14%, 8.15%, and 8.23% (n = 3) for intra-assays and 10.27%, 10.99%, and 11.62% (n = 3) for interassays toward 0.1, 2.0, and 5.0 nM target Kana, respectively, undoubtedly indicating good assay precision and remarkable reproducibility. Additionally, as the photocurrentgeneration tag, the stability of the IO-ZIS/FTO electrode was researched by consecutive on−off light irradiation for 15 cycles during the measuring process (Figure 3C). The photocurrent 7839

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response and shape revealed little fluctuation during 15 cycles (RSD = 1.12%), suggesting good stability of the photocurrent readout. Moreover, as a crucial parameter for the PEC biodetection system, the precision and specificity of the PFMSCA-based IOZIS-PEC platform could further be systematically estimated. Other common antibiotics species of cefixime (CFM), vancomycin hydrochloride (VAN), levofloxacin (LEV), and methicillin (MET) were introduced as the control. In the same manner, these nontarget antibiotics analytes (50 nM, 10-fold concentration of target Kana) were separately measured, and then their mixture with 5.0 nM target Kana was also determined. It shows that the photocurrent response of nontarget analytes including CFM, VAN, LEV, and MET are neglectable, while significant photocurrent responses are observed for the detection of Kana and the mixed solution containing Kana (Figure 3D), thus revealing the satisfactory antijamming capability toward target Kana. From the application perspective, the accuracy of the PFMSCA-based IO-ZIS PEC platform was evaluated by assaying the milk and compared with a commercially Kana-ELISA diagnostic kit. The results are shown in Table 2 (mean ± SD, RSD), and all texp

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01557.

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1 2 3 4 5 6

2.02 2.54 2.98 3.53 3.99 4.98

± ± ± ± ± ±

0.03 0.04 0.06 0.08 0.12 0.16

(1.37%) (1.57%) (2.01%) (2.27%) (3.01%) (3.21%)

Kana ELISA kit 2.04 2.55 2.96 3.50 4.06 4.99

± ± ± ± ± ±

0.04 0.03 0.08 0.09 0.15 0.14

(1.96%) (1.18%) (2.70%) (2.57%) (3.69%) (2.81%)

Material and reagent, apparatus, analysis of milk sample, optimization of experimental conditions (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected]. ORCID

Dianping Tang: 0000-0002-0134-3983 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Grant nos. 21675029 and 21874022) and the Health-Education Joint Research Project of Fujian Province (Grant no. WKJ2016-215).

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method; conc.: mean ± SD (nM, n = 3) PFMSCA-based PEC assay

ASSOCIATED CONTENT

S Supporting Information *

Table 2. Screening of Raw Milk Using the PFMSCA-Based PEC Method and Kana ELISA Kit sample no.

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texp

REFERENCES

(1) Walker, G.; Fraiser, M.; Schram, J.; Little, M.; Nadeau, J.; Malinowski, D. Nucleic Acids Res. 1992, 20, 1691−1696. (2) Zhou, W.; Hu, L.; Ying, L.; Zhao, Z.; Chu, P.; Yu, X. Nat. Commun. 2018, 9, 5012. (3) Reid, M.; Le, X.; Zhang, H. Angew. Chem., Int. Ed. 2018, 57, 11856−11866. (4) Sun, Y.; Sun, Y.; Tian, W.; Liu, C.; Gao, K.; Li, Z. Chem. Sci. 2018, 9, 1344−1351. (5) Zeng, R.; Luo, Z.; Zhang, L.; Tang, D. Anal. Chem. 2018, 90, 12299−12306. (6) Zeng, R.; Su, L.; Luo, Z.; Zhang, L.; Lu, M.; Tang, D. Anal. Chim. Acta 2018, 1038, 21−28. (7) Cui, Y.; Feng, X.; Wang, Y.; Pan, H.; Pan, H.; Kong, D. Chem. Sci. 2019, 10, 2290−2297. (8) Huang, M.; Zhou, X.; Wang, H.; Xing, D. Anal. Chem. 2018, 90, 2193−2200. (9) Chen, J.; Zhou, X.; Ma, Y.; Lin, X.; Dai, Z.; Zou, X. Nucleic Acids Res. 2016, 44, 130. (10) Lu, L.; Jia, H.; Dröge, P.; Li, J. Funct. Integr. Genomics 2007, 7, 221−227. (11) Li, F.; Zhao, H.; Wang, Z.; Wu, Z.; Yang, Z.; Li, C.; Xu, H.; Lyu, J.; Shen, Z. Biosens. Bioelectron. 2017, 91, 692−698. (12) Shen, Z.; Li, F.; Jiang, Y.; Chen, C.; Xu, H.; Li, C.; Yang, Z.; Wu, Z. Anal. Chem. 2018, 90, 3335−3340. (13) Zeng, R.; Luo, Z.; Su, L.; Zhang, L.; Tang, D.; Niessner, R.; Knopp, D. Anal. Chem. 2019, 91, 2447−2454. (14) Xue, Q.; Kong, Y.; Wang, H.; Jiang, W. Chem. Commun. 2017, 53, 10772−10775. (15) Lin, Y.; Zhou, Q.; Tang, D. Anal. Chem. 2017, 89, 11803− 11810. (16) Ren, R.; Cai, G.; Yu, Z.; Tang, D. Sens. Actuators, B 2018, 265, 174−181. (17) Zhuang, J.; Han, B.; Liu, W.; Zhou, J.; Liu, K.; Yang, D.; Tang, D. Biosens. Bioelectron. 2018, 99, 230−236. (18) Wang, S.; Guan, B.; Lou, X. J. Am. Chem. Soc. 2018, 140, 5037− 5040. (19) Cho, W.; Lee, H. J.; Oh, M. J. Am. Chem. Soc. 2008, 130, 16943. (20) Yang, W.; Zhang, L.; Xie, J.; Zhang, X.; Liu, Q.; Yao, T.; Wei, S.; Zhang, Q.; Xie, Y. Angew. Chem., Int. Ed. 2016, 55, 6716.

1.93 1.45 1.87 1.62 1.22 1.37

values were below 2.776 at the 95% confidence level (tcrit[0.05,4] = 2.776) (student’s t test formula).,3436 Hence, there is no obvious difference between the two methods (Kana ELISA kit and PFMSCA-based PEC method), and the accuracy of the PFMSCA-based IO-ZIS-PEC platform is quite satisfactory.

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CONCLUSIONS In summary, this contribution successfully devised a novel PEC platform for the determination of antibiotic residue (Kanamycin, Kana) by coupling IO-ZIS and PFMSCA for exponential signal amplification. Relative to traditional and our previous PEC works,13,31 two overwhelming advantages offered by this research should be highly emphasized. Above all, by highly integrating the Kana-recognition region, template, and palindrome tail into one oligonucleotide in a one-pot mixing program, the complexity of the sensing system is greatly reduced and the sensing operation is simplified. Meanwhile, use of the PFMSCA-assisted liposome-encode assembly and magnetic separation helps in avoiding the sample matrix interference between the substrate and the signal readout. Taking into account the highly efficient and multifunctional properties of the PFMSCA, the concept of palindromic fragment-mediated primer-free provides a new avenue in the design of desirable nucleic acid probes in basic research and convenient biodetection systems. 7840

DOI: 10.1021/acs.analchem.9b01557 Anal. Chem. 2019, 91, 7835−7841

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Analytical Chemistry (21) Wang, P.; Shen, Z.; Xia, Y.; Wang, H.; Zheng, L.; Xi, W.; Zhan, S. Adv. Funct. Mater. 2019, 29, 1807013. (22) Chao, Y.; Zhou, P.; Li, N.; Lai, J.; Yang, Y.; Zhang, Y.; Tang, Y.; Yang, W.; Du, Y.; Su, D.; Tan, Y.; Guo, S. Adv. Mater. 2019, 31, 1807226. (23) Du, C.; Zhang, Q.; Lin, Z.; Yan, B.; Xia, C.; Yang, G. Appl. Catal., B 2019, 248, 193−201. (24) Xiao, B.; Song, S.; Wang, P.; Zhao, Q.; Chuai, M.; Zhang, M. Sens. Actuators, B 2017, 241, 489−497. (25) Wang, S.; Guan, B.; Wang, X.; Lou, X. J. Am. Chem. Soc. 2018, 140, 15145−15148. (26) Yan, A.; Shi, X.; Huang, F.; Fujitsuka, M.; Majima, T. Appl. Catal., B 2019, 250, 163−170. (27) Zhang, G.; Chen, D.; Li, N.; Xu, Q.; Li, H.; He, J.; Lu, J. Appl. Catal., B 2018, 232, 164−174. (28) Li, Q.; Xia, Y.; Yang, C.; Lv, K.; Lei, M.; Li, M. Chem. Eng. J. 2018, 349, 287−296. (29) Yang, M. Q.; Xu, Y. J.; Lu, W.; Zeng, K.; Zhu, H.; Xu, Q. H.; Ho, G. W. Nat. Commun. 2017, 8, 14224. (30) Zeng, R.; Tang, Y.; Zhang, L.; Luo, Z.; Tang, D. J. Mater. Chem. B 2018, 6, 8071−8077. (31) Zeng, R.; Zhang, L.; Su, L.; Luo, Z.; Zhou, Q.; Tang, D. Biosens. Bioelectron. 2019, 133, 100−106. (32) Nguyen, A.; Ma, X.; Park, H.; Sim, S. Sens. Actuators, B 2019, 282, 765−773. (33) Cai, G.; Yu, Z.; Ren, R.; Tang, D. ACS Sens. 2018, 3, 632−639. (34) Luo, Z.; Zhang, L.; Zeng, R.; Su, L.; Tang, D. Anal. Chem. 2018, 90, 9568−9575. (35) Lv, S.; Zhang, K.; Zeng, Y.; Tang, D. Anal. Chem. 2018, 90, 7086−7093. (36) Qiu, Z.; Shu, J.; Liu, J.; Tang, D. Anal. Chem. 2019, 91, 1260− 1268.

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DOI: 10.1021/acs.analchem.9b01557 Anal. Chem. 2019, 91, 7835−7841