Quantitative Label-Free Listeria Analysis Based On ... - ACS Publications

Letter pubs.acs.org/acssensors

Quantitative Label-Free Listeria Analysis Based On Aptamer Modified Nanoporous Sensor Chun-Xia Zhou,‡ Ri-Jian Mo,‡ Zhi-Meng Chen,‡ Juan Wang, Guo-Zhu Shen, Yi-Ping Li, Qin-Guo Quan, Ying Liu, and Cheng-Yong Li* Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, P. R. China S Supporting Information *

ABSTRACT: In this Letter, we proposed a nanoporous sensor to realize a quick and sensitive detection of Listeria monocytogenes with the application of porous anodic aluminum oxide membrane combined with a specific aptamer. On the basis of electrochemical detection, it can be known that the reduction current response upon the diffusion flux of electrochemical probe is affected by the charge repulsion and steric blockage of Listeria monocytogenes. A rapid bacteria detection can be achieved within 10 min, with a high sensitivity of 100 CFU/mL and a good linear range between 100 to1250 CFU/mL. Moreover, it has a high specificity for Listeria monocytogenes even exposed in 108 CFU/mL Staphylococcus aureus or Escherichia coli. KEYWORDS: aptamer, Listeria monocytogenes, steric blockage, charge repulsion, electrochemical detection, anodic aluminum oxide

B

Currently, a novel scheme for highly sensitive analysis of biomolecules was proposed by Gyurcsányi et al. based on probe ion diffusion flux using nanoporous membranes.9,10 This scheme has been extensively applied in biochemical analysis.11−17 Ding et al. proposed single-molecule biosensors based on an aptamer-encoded glass nanopore.18 Li et al. built a nanoporous membrane sensor and applied it in quantitative label-free DNA analysis.19 Gao et al. used morpholino functionalized nanoporous membrane for single nucleotide polymorphisms detection.20 In our previous work, we developed a simple label-free electrochemical biosensor, which can be used to detect lead ions using a nanoporous membrane modified with DNA and morpholino.21 It was based on the charge effect or steric blockage of nanochannels inserted in membranes. Nanoporous membranes have also been successfully applied in bacteria detection. A nanoporous membrane impedimetric immunosensor was proposed by Joung et al. and the trace bacterial pathogens in whole milk can be detected.22 A new sensing strategy was proposed by Tang et al. using silicon nanopore array via indirect Fourier transformed reflectometric interference spectroscopy and the target bacteria were quickly detected based on its blockage effect.23 The analyzing principle of bacteria is different from that of biomolecules. Usually, bacteria cannot enter nanochannels due to their larger size compared with the

acterial foodborne pathogens are a type of serious health hazard for people all over the word, especially when handheld or portable devices are not sufficient, so that pathogenic bacteria can be quickly detected with high sensitivity and selectivity. As one of the most virulent foodborne pathogens, Listeria monocytogenes (LM) can cause death in 20−30% of clinical infections.1 It is widely distributed in such environmental mediums as water, sediment, and seafood. In addition, LM is able to survive in an environment with high concentrations of salt at temperatures 3−45 °C and pHs 5.4−9.6.2,3 The existence of LM in aquatic environments polluted by urban runoff and effluent from public water treatment plants threatens human health. Thus, a broad consensus is reached regarding the need for sensitive detection of LM in the aquatic environment for protection of human health. Traditional methods, including biochemical tests, cell culture, microscopy, and luminescence, require a large expenditure of labor and time (generally 24−48 h).4 Some newly developed molecular detection techniques, such as PCR and ELISA, have been put into application.5−8 Such new techniques can achieve significantly decreased detection time as compared with conventional techniques, although they still need several hours for identification of the target pathogens and are unable to detect bacteria in real time. On the other hand, the newly developed techniques require the involvement of complex and expensive instruments and highly trained personnel, and are not operating smoothly with routine screening. © XXXX American Chemical Society

Received: May 18, 2016 Accepted: August 2, 2016

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DOI: 10.1021/acssensors.6b00333 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors

time was 0.5 h. Then the sample was immersed in a mixed acid containing 1.5 wt % chromic acid and 5 wt % phosphoric acid for 30 min to remove alumina. The condition of the first anodization was the same as the pre-anodization process. The membrane was then treated with hydroxylation reaction in boiled 30% H2O2 for 0.5 h and dried in N2. After that, the membrane was treated with 10% 3-aminopropyltrimethoxysilane (APTMS) solution (diluted with acetone) for 12 h. Subsequently, the membrane was washed and then cured at 120 °C for 2 h and APTMS was cross-linked to form a stable silane layer. The condition of the second anodization was the same as the first one, but the anodization time was extended to 20 h. After implementation of the second anodization, a fresh alumina layer was generated. Finally, it was immersed in SnCl2 solution to remove the alumina substrate, and then immersed in 5 wt % phosphoric acid to carry out the bottom pore-opening step. AAO membrane with 100 nm thickness was fabricated in 0.3 M oxalic acid solution under 40 V. AAO membrane with 200 nm thickness was prepared in 0.3 M phosphoric acid solution under 130 V. After being sputtered with Au, AAO membrane was placed into the homemade cell which contains 2 mL KCl electrolyte (1 mM). Then, 20 μL of aptamer solution (100 μM prepared in 100 mM PBS, pH 8.0) was dropped on the surface of AAO membrane without Au film and left in an Ar glovebox overnight. The excessive aptamer was washed away using PBS solution. Aptamer sequence: 5′-CHO-ATC CAT GGG GCG GAG ATG AGG GGG AGG AGG GCG GGT ACC CGG TTG AT-3′. The electrochemical setup is shown in Figure S1. A different concentration of LM solution was introduced into the cell containing 1 mM KCl electrolyte and then it was stirred slowly. Bacterial broth and nontarget bacteria (other bacteria may exist in LM) were washed off from the membrane by 1 mM KCl solution. AAO membrane with Au coating, Pt electrode, and Ag/AgCl electrode constituted a three-electrode system. The flux of Fe(CN)63− crossing the nanoporous sensor chip was measured by the cathodic reduction current at 0 V. We investigated the AAO membranes of different sizes (20, 100, and 200 nm) in the hope of selecting one for realizing the optimal performance of the sensor. According to Figure S2, we can see that when the AAO membrane is 20 nm, the current change (Δi) is the maximum value (170 nA, Δi/i = 69.4%); when the AAO membrane is 100 nm, Δi is 107 nA (Δi/i = 35.3%); when the AAO membrane is 200 nm, Δi is 62 nA (Δi/i = 17.6%). Based on these results, it can be concluded that the AAO membrane of 20 nm can combine more LM, because the 20 nm AAO membrane has the biggest specific area, and thus the density of aptamer on the surface of 20 nm AAO membrane is the highest. Therefore, 20 nm AAO membranes were used for the later detection experiments. Figure 1 shows the characteristics of AAO membrane prepared in sulfuric acid. As shown in Figure 1a, a well-ordered array of nanopores (pore diameter is about 20 nm) can be observed. The thickness of the whole AAO membrane is about 20 μm (Figure 1b). Fluorescein isothiocyanate (FITC) is selectively linked on the surface or nanochannels near the surface, which was modified with APTMS. According to the laser scanning confocal microscopy (LSCM) image of the AAO membrane cross section in Figure 1b, green florescence from the top layer can be clearly observed, which is relatively stronger than that of the lower layer. Fourier transform infrared spectra (FTIR) were used to monitor the modification process

nanochannels. The reaction between the bacteria and the aptamer occurs on the surface of the nanoporous membrane. In this case, the aptamer modified in nanochannels cannot react with bacteria on the surface. It would increase the unstable factors of nanoporous membranes and the background signal is unstable. First, chemical modification (e.g., coupling reagent) in nanochannels is difficult, especially when the nanochannel size is smaller than 20 nm. Second, the aptamer has difficulty entering into nanochannels to react with coupling reagent when its sequence is too long. In this work, a nanoporous sensor was proposed for rapid and sensitive detection of LM based on a porous anodic aluminum oxide (AAO) membrane modified with a specific aptamer. In order to reduce the negative effects caused by chemical modification in nanochannels, an in situ chemical modification method was used in our experiments, and only the surface or nanochannels near the surface of the AAO membranes was modified with aptamer. It could achieve rapid bacteria detection within 10 min with a high sensitivity of 100 CFU/mL. In addition, it has a high specificity for LM. Scheme 1 shows the detection mechanism of LM. After being introduced into the feeding cell, LM will be attracted to Scheme 1. Scheme of Nanoporous Sensor in Detection of LM

the surface of the nanoporous membrane via the aptamer specific binding. LM is about 2 μm in length and 0.5 μm in width and nanoporous membranes would be blocked. In addition, LM cells have negative charge, −14.2 mV, as their cell wall consists of phosphate and carboxylate groups.24 Due to the adhesion of LM, the surface charge of the AAO membrane will be increased significantly, and the electrochemical probe Fe(CN)63− will be repelled. A slow Fe(CN)63− flux and weak reduction current can be observed under the influences of steric blockage and charge repulsion of LM. A reduction potential of 0 V is used for the detect of Fe(CN)63−; in this process, Fe(CN)63− electrochemical reduction is subjected to diffusion control. Reduction current is proportional to Fe(CN)63− concentration. Therefore, we can determine Fe(CN)63−diffusion flux across the nanoporous membrane by observing the steady-state reduction current (i), and conduct quantitative detection of LM by regarding this reduction current as an analytical signal. Using a two-step anodic oxidation technique, AAO membranes with different sizes were prepared.11,25−27 Taking the preparation process of 20 nm AAO membrane as an example, a pre-anodization step was first conducted to gain the finely organized nanochannels before the two-step anodization process was performed. The pre-anodization process was anodized in 0.2 M sulfuric acid at 20 V. The pre-anodization B

DOI: 10.1021/acssensors.6b00333 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors

As Fe(CN)63− passed through the nanoporous membranes, a larger steady-state current response can be observed. As shown in Figure 2a, as the concentration of LM increases from 100 to

Figure 1. Characteristics of AAO membranes. (a) SEM image of the surface of AAO membrane as prepared. (b) Cross section SEM and LSCM (inserted) images of cross section of the AAO membrane as prepared.

Figure 2. Electrochemical detection results. (a) Current response curves of the nanoporous sensor in the presence of various concentrations LM and the LM density ranging from 100 to 3000 CFU/mL. (b) Change of current responses (Δi) versus LM concentration (n = 5).

(Figure S3). However, there is no obvious difference between pure AAO membrane and the APTMS modified one due to the extremely small quantity of comparative samples. In addition, FTIR spectra are the same in the pure AAO membrane and after aptamer modification. In order to confirm the successful modification of APTMS and aptamer on the surface of the AAO membrane, X-ray photoelectron spectroscopy (XPS) was carried out (Figures S4, S5). The nonmodified AAO membrane showed neither N 1s nor P 2p peak. After the membrane was modified with APTMS, an obvious peak of nitrogen can be observed (the phosphate peak was remained invisible). After being treated with aptamer, the peak of N 1s is significantly increased because of the mass of nitrogen in the DNA aptamer. Meanwhile, the peak of P 2p appears due to the phosphate in the backbone structure of DNA aptamer. The presence or absence of aptamer on the surface of the AAO membrane was also detected by the electrochemical method (Figure S6). The current is decreased slightly after the aptamer was modified due to the increase of electrostatic repulsion, which is in accord with the references.19,20 The incubation time after LM was added to the feeding cell was investigated. As shown in Figure S7, the current (Δi) decreases at a sharp rate for 2−6 min and then the decreasing rate becomes gradually flat from 8 min. After 10 min, Δi reaches the steady state. Therefore, 10 min is an optimal incubation time, which was adopted as a standard for the subsequent experiments.

3000 CFU/mL, the steady-state current of Fe(CN)63− shows a gradual decreasing trend. These results indicate that there are more LM captured on the surface of AAO membranes as the LM concentration increased. It can be observed that the current shows a decreasing trend due to LM addition, and such a decreasing trend is linearly proportional to LM concentration. The linear regression equation between current change and LM concentration is Δi (nA) = 0.63 + 0.16C (CFU/mL) and R2 is 0.999. It can be seen from Figure 2b that the detection limit of LM is 100 CFU/mL. Selectivity is very important in detection. Base on two other common nontarget bacteria such as Staphylococcus aureus (SA) and Escherichia coli (E. coli), the selectivity for LM was successfully evaluated. Figure 3 shows the current response for the nanoporous membrane after adding 108 CFU/mL SA or E. coli, respectively. The current is decreased; however, the decrease rate is not obvious, which may be caused by the nonspecific absorption of SA or E. coli. Results show that such a proposed nanoporous sensor shows highly selective recognition for LM only. In order to determine the selectivity of the proposed nanoporous sensor, it was immersed in a sample solution containing 100, 103, 3 × 103 CFU/mL of LM or 108 CFU/mL nontarget bacteria (SA and E. coli) for 10 min, and then it was examined via SEM. It can be seen from Figure 4 that only LM can be captured on the surface of the nanoporous sensor chip. In addition, it can be clearly seen that Fe(CN)63− diffusion flux C

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that the nanoporous sensor could effectively identify LM in water samples. Table 1. Results of the Determination of LM in Water Samples from Different Fish Markets (n = 3) sample Sample Sample Sample Sample Sample

nanoporous sensor (CFU/mL) 1 2 3 4 5

108 420 452 643 895

± ± ± ± ±

4 3 4 5 4

SPC (CFU/mL) 110 417 456 638 902

± ± ± ± ±

5 4 5 5 3

In summary, we have developed a novel nanoporous sensor for rapid and sensitive detection of LM. It is an electrochemical method using AAO membrane modified with specific aptamer. It can be concluded that the origin of current response from the electrochemical probe diffusion flux is affected by charge repulsion and steric blockage of LM. With the application of the proposed sensor, rapid detection of bacteria can be completed within 10 min, which achieved a high sensitivity of 100 CFU/mL and a good linear range between 100 and 1250 CFU/mL. In addition, it has a high specificity even exposed to 108 CFU/mL SA or E. coli. It may selectively capture bacteria in a sample with multiple kinds of bacteria by tuning the pore size and using different aptamer recognition molecules. The proposed sensing strategy shows promising application prospects for quantitative label-free analysis of LM in food, environmental samples, and other medical diagnostics.

Figure 3. Selectivity tests of the novel nanoporous sensor. (a) Curves of current response measured for E. coli, SA, and LM. (b) Change of current responses (Δi) calculated from (a) (n = 5).

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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.6b00333. Cell assembly and electrochemical measurements, SEM images, FTIR spectra, XPS, and current response curves (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-759-2396270. Fax: +86759-2396270. Author Contributions ‡

Chun-Xia Zhou, Ri-Jian Mo, and Zhi-Meng Chen contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (21405024), Science and Technology Planning Project of Guangdong Province (2014A020212683, 2016A020210114), Science and Technology Planning Project of Zhanjiang City (2015A03025), Foundation for Outstanding Young Teachers of Guangdong Ocean University (2014005), Training Programs of Innovation and Entrepreneurship for Undergraduates (201510566003).

Figure 4. Surface SEM images of the AAO membrane after immersion into different bacteria solutions. (a) Without bacteria. (b) 100 CFU/ mL LM. (c) 103 CFU/mL LM. (d) 3 × 103 CFU/mL LM. (e) 108 CFU/mL E. coli. (f) 108 CFU/mL SA.

through the AAO membrane is significantly blocked due to the existence of LM, and thus the current response is reduced. The water samples collected from different fish markets were analyzed using the nanoporous sensor. The values of LM in water samples were consistent with those found by the standard plate count method (SPC) (Table 1). These results indicate

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REFERENCES

(1) Boehm, A. B.; Yamahara, K. M.; Love, D. C.; Peterson, B. M.; McNeill, K.; Nelson, K. L. Covariation and Photoinactivation of

D

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Single Nucleotide Polymorphisms Detection. Anal. Chem. 2015, 87, 3936−3941. (21) Chen, Z. M.; Shen, G. Z.; Li, Y. P.; Zhang, P.; Ji, H. W.; Liu, S. C.; Li, C. Y.; Qian, Z. J. A Novel Biomimetic Logic Gate for Sensitive and Selective Detection of Pb(II) Base on Porous Alumina Nanochannels. Electrochem. Commun. 2015, 60, 83−87. (22) Joung, C. K.; Kim, H. N.; Lim, M. C.; Jeon, T. J.; Kim, H. Y.; Kim, Y. R. A Nanoporous Membrane-Based Impedimetric Immunosensor for Label-Free Detection of Pathogenic Bacteria in Whole Milk. Biosens. Bioelectron. 2013, 44, 210−215. (23) Tang, Y.; Li, Z.; Luo, Q.; Liu, J.; Wu, J. Bacteria Detection Based on its Blockage Effect on Silicon Nanopore Array. Biosens. Bioelectron. 2016, 79, 715−720. (24) Briandet, R.; Meylheuc, T.; Maher, C.; Bellon-Fontaine, M. N. Listeria monocytogenes Scott A: Cell Surface Charge, Hydrophobicity, and Electron Donor and Acceptor Characteristics under Different Environmental Growth Conditions. Appl. Environ. Microb. 1999, 65, 5328−5333. (25) Li, C. Y.; He, L. Fabrication of CNx/C Nanotube Arrays via Chemical Modification in Situ and application in an Electronic NanoDevice. RSC Adv. 2014, 4, 6031−6034. (26) Masuda, H.; Fukuda, K. Ordered Metal Nanohole Arrays Made by a Two-Step Replication of Honeycomb Structures of Anodic Alumina. Science 1995, 268, 1466−1468. (27) Chen, W.; Wu, J. S.; Xia, X. H. Porous Anodic Alumina with Continuously Manipulated Pore/Cell Size. ACS Nano 2008, 2, 959− 965.

Traditional and Novel Indicator Organisms and Human Viruses at a Sewage-Impacted Marine Beach. Environ. Sci. Technol. 2009, 43, 8046−8052. (2) Lu, X.; Rasco, B. A.; Kang, D. H.; Jabal, J. M. F.; Aston, D. E.; Konkel, M. E. Infrared and Raman Spectroscopic Studies of the Antimicrobial Effects of Garlic Concentrates and Diallyl Constituents on Foodborne Pathogens. Anal. Chem. 2011, 83, 4137−4146. (3) EL-SHENAWY, M. A.; EL-SHENAWY, M. A. Listeria spp. in the coastal environment of the Aqaba Gulf, Suez Gulf and the Red Sea. Epidemiol. Infect. 2006, 134, 752−757. (4) Jadhav, S.; Bhave, M.; Palombo, E. A. Methods used for the detection and subtyping of Listeria monocytogenes. J. Microbiol. Methods 2012, 88, 327−341. (5) Liu, Z.; Zhu, J.; Yang, C.; Li, X. Visual detection of Listeria monocytogenes using unmodified gold nanoparticles based on a novel marker. Anal. Methods 2015, 7, 8159−8164. (6) Wang, R.; Ruan, C.; Kanayeva, D.; Lassiter, K.; Li, Y. TiO2 Nanowire Bundle Microelectrode Based Impedance Immunosensor for Rapid and Sensitive Detection of Listeria monocytogenes. Nano Lett. 2008, 8, 2625−2631. (7) Beauchamp, S.; D’Auria, S.; Pennacchio, A.; Lacroix, M. A new competitive fluorescence immunoassay for detection of Listeria monocytogenes. Anal. Methods 2012, 4, 4187−4192. (8) Chen, R.; Huang, X.; Xu, H.; Xiong, Y.; Li, Y. Plasmonic EnzymeLinked Immunosorbent Assay Using Nanospherical Brushes as a Catalase Container for Colorimetric Detection of Ultralow Concentrations of Listeria monocytogenes. ACS Appl. Mater. Interfaces 2015, 7, 28632−28639. (9) Gyurcsanyi, R. E.; Vigassy, T.; Pretsch, E. BiorecognitionModulated Ion Fluxes through Functionalized Gold Nanotubules as a Novel Label-Free Biosensing Approach. Chem. Commun. 2003, 2560− 2561. (10) Jágerszki, G.; Gyurcsányi, R. E.; Höfler, L.; Pretsch, E. Hybridization-Modulated Ion Fluxes through Peptide-Nucleic-AcidFunctionalized Gold Nanotubes. A New Approach to Quantitative Label-Free DNA Analysis. Nano Lett. 2007, 7, 1609−1612. (11) Li, C. Y.; Ma, F. X.; Wu, Z. Q.; Gao, H. L.; Shao, W. T.; Wang, K.; Xia, X. H. Solution-pH-Modulated Rectification of Ionic Current in Highly Ordered Nanochannel Arrays Patterned with Chemical Functional Groups at Designed Positions. Adv. Funct. Mater. 2013, 23, 3836−3844. (12) Li, C. Y.; Tian, Y. W.; Shao, W. T.; Yuan, C. G.; Wang, K.; Xia, X. H. Solution pH Regulating Mass Transport in Highly Ordered Nanopore Array Electrode. Electrochem. Commun. 2014, 42, 1−5. (13) Wang, C.; Fu, Q.; Wang, X.; Kong, D.; Sheng, Q.; Wang, Y.; Chen, Q.; Xue, J. Atomic Layer Deposition Modified Track-Etched Conical Nanochannels for Protein Sensing. Anal. Chem. 2015, 87, 8227−8233. (14) Gu, Z.; Ying, Y. L.; Cao, C.; He, P.; Long, Y. T. Accurate Data Process for Nanopore Analysis. Anal. Chem. 2015, 87, 907−913. (15) Yu, J.; Luo, P.; Xin, C.; Cao, X.; Zhang, Y.; Liu, S. Quantitative Evaluation of Biological Reaction Kinetics in Confined Nanospaces. Anal. Chem. 2014, 86, 8129−8135. (16) Wang, H.; Hou, S.; Wang, Q.; Wang, Z.; Fan, X.; Zhai, J. DualResponse for Hg2+ and Ag+ ions Based on Biomimetic Funnel-Shaped Alumina Nanochannels. J. Mater. Chem. B 2015, 3, 1699−1705. (17) Sun, Z.; Zhang, F.; Zhang, X.; Tian, D.; Jiang, L.; Li, H. Chiral Recognition of Arg Based on Label-Free PET Nanochannel. Chem. Commun. 2015, 51, 4823−4826. (18) Ding, S.; Gao, C. L.; Gu, L. Q. Capturing Single Molecules of Immunoglobulin and Ricin with an Aptamer-Encoded Glass Nanopore. Anal. Chem. 2009, 81, 6649−6655. (19) Li, S. J.; Li, J.; Wang, K.; Wang, C.; Xu, J. J.; Chen, H. Y.; Xia, X. H.; Huo, Q. A Nanochannel Array-Based Electrochemical Device for Quantitative Label-free DNA Analysis. ACS Nano 2010, 4, 6417− 6424. (20) Gao, H. L.; Wang, M.; Wu, Z. Q.; Wang, C.; Wang, K.; Xia, X. H. Morpholino-Functionalized Nanochannel Array for Label-Free E

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