Peptide-Assembled Graphene Oxide as a ... - ACS Publications

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Peptide-Assembled Graphene Oxide as a Fluorescent Turn-On Sensor for Lipopolysaccharide (Endotoxin) Detection Seng Koon Lim,† Peng Chen,† Fook Loy Lee,† Shabbir Moochhala,†,‡ and Bo Liedberg*,† †

Centre for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Drive, 637553, Singapore ‡ DSO National Laboratories, 27 Medical Drive #09-01, 117510, Singapore

Downloaded by RUTGERS UNIV on September 1, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.analchem.5b02270

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ABSTRACT: Lipopolysaccharide (LPS) is a toxic inflammatory stimulator released from the outer cell membrane of Gram-negative bacteria, known to be directly related to, for example, septic shock, that causes millions of casualties annually. This number could potentially be lowered significantly if specific, sensitive, and more simply applicable LPS biosensors existed. In this work, we present a facile, sensitive and selective LPS sensor, developed by assembling tetramethylrhodamine-labeled LPSbinding peptides on graphene oxide (GO). The fluorescence of the dyelabeled peptide is quenched upon interaction with GO. Specific binding to LPS triggers the release of the peptide-LPS complex from GO, resulting in fluorescence recovery. This fluorescent turn-on sensor offers an estimated limit of detection of 130 pM, which is the lowest ever reported among all synthetic LPS sensors to date. Importantly, this sensor is applicable for detection of LPS in commonly used clinical injectable fluids, and it enables selective detection of LPS from different bacterial strains as well as LPS on the membrane of living E. coli.

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another peptide-functionalized polydiacetylene liposome, functioning as a fluorescent turn-on sensor, was reported.13 More recently, gold nanoparticle-based14 and copolythiophenebased15 LPS sensors have been proposed, with significant improvement of the estimated detection limit down to low nanomolar and subnanomolar levels, respectively. However, these sensors are still not robust and sensitive enough to meet the requirement to detect LPS in the picomolar regime. Graphene oxide (GO), which is an oxidized version of graphene, has become a promising material for biotechnology and biosensing application, because of its superior characteristics, such as large surface area, good water solubility, biocompatibility, easy surface modification, low manufacturing cost, etc.16,17 GO also displays very high fluorescence quenching efficiency,18 which has been explored extensively for applications in Raman spectroscopy,19 biological imaging,20 biosensing,21,22 etc. Several GO-based fluorescence turn-on biosensors have been reported for the detection of DNA,23−27 metal ions,28,29 ATP,30,31 protease,32−34 proteins,33,35,36 and other chemicals37−39 using a labeled ssDNA, PNA, or protease substrate peptide, assembled or conjugated with GO. The aforementioned fluorescence turn-on sensors can be categorized into three types. First, the binding of target molecules to the dye-labeled receptor induces a change in either the

ipopolysaccharide (LPS), or endotoxin, is a major component in the outer cell membrane of Gram-negative bacteria. It is a glycolipid comprising a variable polysaccharide domain attached to a conserved glucosamine-based phospholipid called lipid A (Scheme 1).1 It is a very powerful and toxic inflammatory stimulator that induces host monocytes and macrophages to secrete a wide range of inflammatory cytokines, including interleukins-1, tumor necrosis factor-α, and interleukin-8.2,3 Excessive secretion of these cytokines may contribute to organ failure, or result in sepsis or septic shock, which is a significant medical problem affecting ∼700 000 patients and causing 250 000 casualties annually in the United States alone.4,5 In Europe, septic shock is one of the main causes of death in hospitals (mortality rate of 30%−50%).2 The development of specific and sensitive LPS biosensors is urgently required. The currently used enzymatic Limulus amebocyte lysate (LAL) assay is highly susceptible to changes in temperature and pH,6 various interfering factors in the samples,7−9 and requires cumbersome sample preparation, storage, and controlled experimental conditions.6,10 Therefore, in recent years, numerous efforts have been devoted to the development of alternative synthetic sensor concepts for LPS. The first reported synthetic sensor for LPS was based on functionalized polydiacetylene liposomes, which only could detect high LPS concentration above 100 μM.11 Another FRET-based sensor enabled LPS detection down to micromolar concentrations by using a peptide hairpin beacon derived from the LPS-binding domain of the LPS receptor CD14.12 Yet © XXXX American Chemical Society

Received: June 16, 2015 Accepted: August 24, 2015

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DOI: 10.1021/acs.analchem.5b02270 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry


Scheme 1. (A) Structure of LPS and (B) Sensing Principle of the Peptide−GO-Assembled LPS Sensor, Including Attachment of Fluorescence Labelled Peptide on GO and Subsequent Detachment of the Peptide Triggered by LPS

conformation or orientation of the receptor, which, in turn, induces a change in the distance between the graphene surface and the dye, and, ultimately, the fluorescence emission. Second, enzymatic digestion of dye-labeled substrates immobilized onto the graphene surface release the dye into solution. Third, a dyelabeled receptor is assembled onto the graphene surface through electrostatic/π−π interaction, and the competitive binding from target to receptor release the dye-labeled receptor from the graphene surface. Among those, the third type of fluorescence turn-on sensor provides a general platform for detection of bimolecular targets. Herein, we present the first GO-based fluorescence turn-on biosensor employing an allsynthetic peptide receptor for the detection of LPS based on a binding-triggered release strategy. This assay offers highly sensitive and specific LPS detection, down to the picomolar level. We further explored the opportunity to employ the developed assay format for the detection of LPS spiked in clinically relevant matrices. We demonstrate herein the feasibility of the assay for three injectable fluids/supplements. The principle of this biosensor is shown in Scheme 1. Briefly, a synthetic LPS-binding peptide (KC-13, sequence: KKNYSSSISSIHC) was selected using the phage-display method.40 The pI of the peptide is 2, giving the peptide a net charge of +2 at pH 7. Cysteine at the C-terminal of peptide allows us to attach tetramethylrhodamine (TMRho) dye through the reaction of thiol with maleimide to form the fluoropeptide KC-13R. Together with the TMRho dye, the fluoropeptide will physically adsorb to the negatively charged GO via electrostatic interactions and/or π−π stacking in solution, resulting in fluorescence quenching. The fluorescence signal recovers when LPS, a negatively charged glycolipid, competitively binds to the peptide, releasing dye-labeled peptide from the GO sheets.

Aldrich and were of analytical grade. Lipopolysaccharides from Escherichia coli 0111:B4, Pseudomonas aeruginosa 10, Salmonella typhosa, and Klebsiella pneumonia were purchased from Sigma− Aldrich as lyophilized powders. The molecular weight of commercial LPS varies between 4 and 20 kDa. In our calculations, we assumed a molecular weight of 10 kDa. Clinical-grade 0.9% sodium chloride intravenous infusion BP and compound sodium lactate intravenous infusion BP (Hartmann’s solution) were purchased from B. Braun. Insulin aspart (NovoRapid FlexPen) was purchased from Novo Nordisk. To prepare the peptide−GO assembly, 5 μM of TMRholabeled peptide in PBS, pH 7.4 was mixed with a 22 μg/mL GO solution. GO was synthesized using a modified Hummer’s method. Raman and fluorescence quenching experiments were performed to check the assembling of peptide onto GO. The peptide−GO stock solution was sonicated for 15 min before the experiments were carried out. For LPS sensing experiments, LPS was dissolved in deionized (DI) water with a resistivity of 18 MΩ at 10 μM, 100 nM, and 1 nM stock concentrations. The LPS stock solution was vortexed and warmed to 42 °C before sensing experiments to avoid micelles/aggregates formation. Different concentrations of the LPS were added to the GO−peptide assembly in PBS buffer, and fluorescence spectra of each sample was then recorded with λex/λem = 480 nm/572 nm at 25 °C. For LPS sensing in real samples/matrices, clinical-grade 0.9% sodium chloride intravenous infusion BP, compound sodium lactate intravenous infusion BP (Hartmann’s solution), insulin aspart (NovoRapid FlexPen) were spiked with 2, 4, and 6 μM of LPS for LPS detection. The fluorescence responses were compared with LPS-spiked PBS buffer. All fluorescence measurements were performed using a Fluorolog-3 spectrofluorometer (Jobin Yvon). Raman measurements were conducted with a WITech alpha 300 confocal Raman spectrometer, with a 488 nm laser. The Raman peak of Si at 520 cm−1 was used as reference. Scanning electron microscopy (SEM) measurement was made on a JEOL Model 7600F SEM system. Atomic force microscopy (AFM) measurement was carried out with a Dimension 3100 AFM microscope in tapping mode with a Si tip (Veeco; spring constant = 42 N m−1; resonance frequency = 320 kHz) under ambient conditions.

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EXPERIMENTAL SECTION KC-13 (sequence: KKNYSSSISSIHC) and scrambled peptide (sequence: YISKSNSSKIHSC) were synthesized commercially by GL Biochem (Shanghai, China), purified by reverse-phase high-performance liquid chromatography (HPLC), and checked by mass spectroscopy. KC-13R and scrambled fluoropeptide was synthesized by reacting 1 mM peptide with a 5-fold molar excess of tetramethyrhodamine-5-maleimide via thiol-maleimide reaction for 2 h. Fluorescent-labeled peptide was purified by HPLC and checked by thin layer chromatography on silica gel plates. 1-Palmitoyl-2-oleoyl-sn-glycero-3phospho-(1′-rac-glycerol) (POPG) was purchased from Avanti Polar Lipids. All other chemicals were purchased from Sigma− B


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RESULTS AND DISCUSSION GO was synthesized using a modified Hummer’s method41,42 and characterized by SEM and AFM (see Figure S1 in the Supporting Information). The thickness of as-synthesized GO sheets measured via AFM is ∼0.97 nm. The adsorption of KC13R onto GO is confirmed by a blue shift of the G-band from 1580 cm−1 to 1588 cm−1 in the Raman spectra (Figure 1A).

Figure 2. (A) Fluorescence titration spectra of peptide−GO assembly upon addition of 0 to 10 μM of LPS. The inset shows a photograph of the corresponding color change in the absence (left) and presence (right) of 8 μM LPS. (B) Calibration curve of fluorescence intensity change [(F/F0) − 1] vs the concentration of LPS (0−20 nM). F and F0 are fluorescence intensities at 572 nm with and without LPS, respectively.

Figure 1. (A) Raman scattering of GO with and without KC-13R peptide. (B) Fluorescence emission spectra of 5 μM KC-13R peptide in the presence of various concentrations of GO.

concentration ranging from 0 to 20 nM (Figure 2B). Notably, the sensing response saturates within 5 min, which is much faster than for the LAL method (∼1 h). More importantly, the sensor remains stable at 25 °C, without special storage and stringent temperature requirement. The limit of detection (LOD) is calculated using the standard equation as reported, LOD = 3Q/S, where Q is the standard deviation of blank solution obtained in three serial measurements, and S is the slope of the calibration curve.15,43 The LOD is calculated to be equal to 126.1 pM or 130 pM. This LOD is significantly improved, compared to previously reported values, and is the lowest ever reported for all synthetic LPS sensors.11−13,15,44 Furthermore, in the high concentration regime (e.g., the presence of 8 μM LPS), the response can be easily distinguished by the naked eye under UV light (see inset of Figure 2A). The selectivity of the LPS biosensor was evaluated by monitoring the fluorescence recovery response of the GO− peptide assembly in the presence of several potential coexisting interferences in buffer solution. As shown in Figure 3, all of the molecules known to affect the LAL assay do not produce a significant increase/decrease in the fluorescence intensity ratio (F/F0) (black columns), including ethylenediaminetetraacetic acid (EDTA, a chelating agent known to inhibit LAL assay), citrate (anions), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′rac-glycerol) (POPG, an anionic phospholipid from bacterial membrane), and phosphate and glucose (functional groups found in LPS). In contrast, the addition of 10 μM LPS induces changes in the F/F0 ratio by at least a factor of 3. We further performed LPS sensing experiments in samples containing 2,

Moreover, Figure 1B shows the fluorescence emission spectra of 5 μM KC-13R peptide in the presence of various concentrations of GO. Without GO, the solution of the peptide KC-13R exhibits a strong fluorescence. Upon addition of GO, however, the fluorescence intensity of KC-13R decreases as the concentration of GO increases. More than 95% of the initial fluorescence is instantly quenched in the presence of 22 μg/mL GO, indicating that the fluorescence of the peptide bound to GO can be quenched very efficiently. The binding between GO and the peptide equilibrates within 5 min and remains stable for at least 24 h. Figure 2A shows the fluorescence recovery of KC-13R with different concentrations (2 nM to 10 μM) of LPS extracted from Escherichia coli. To validate that LPS itself does not induce fluorescence changes of KC-13R, a control experiment titrating LPS with KC-13R solutions in the absence of GO was performed. No obvious fluorescence change was induced by LPS in the sample without GO (see Figure S2 in the Supporting Information). Another control experiment replacing KC-13R with a scrambled peptide also gives marginal fluorescence increase, suggesting the role of sequence-specific KC-13R peptide as the LPS receptor in the biosensor (see Figure S2). These results indicate that the specific binding of LPS to the TMRho-labeled KC-13R peptide results in desorption of the peptide (or the peptide−LPS complex) from the surface of GO. Plotting the normalized fluorescence (F/F0) − 1, where F0 is the fluorescence intensity without LPS versus the LPS concentration reveals a linear relationship (correlation coefficient of R2 = 0.99043) with the LPS C


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adding 2 μM purified LPS from four common Gram-negative human pathogens, namely, Escherichia coli, Klebsiella pneumonia, Samonella thyphosa, and Pseudomonas aeruginosa. Interestingly, LPS from these bacterial strains gives similar F/F0 response, except for LPS from P. aeruginosa, a common opportunistic human pathogen, which gives a significantly higher response (Figure 4). The increased response for P. aeruginosa could be due, in part, to higher binding affinity and/or binding capacity to KC-13 peptide.


Figure 3. Fluorescence intensity ratio (F/F0) of the peptide−GO sensor in the presence of various potential interferents. BSA used was 1 mg/L and the other molecules used were 20 μM. Gray columns and black columns refer to fluorescence increase/decrease for these interferents with and without 10 μM LPS, respectively. F and F0 are fluorescence intensities at 572 nm with and without interferents and LPS, respectively.

20, and 200 μM of glucose, EDTA, phosphate or citrate, and found minimal false negatives/positives and very small enhancement/inhibitory effects for our LPS biosensor (Figure S3 in the Supporting Information). In summary, our results show that the assay is highly selective to LPS, even in the presence of potential interferents. The LAL assay often suffers from unwanted interferences caused by the matrix effects of real samples,7−9 which may lead to poor precision, as well as false negative/positive results. To check whether this assay can be used to detect LPS in real samples, we tested it with three injectable clinical solutions of increasing complexity, which require a reliable and sensitive endotoxin test (to ensure the absence of LPS/endotoxin) before being injected into a patient. Briefly, clinical-grade sodium chloride intravenous infusion, compound sodium lactate intravenous infusion (Hartmann’s solution), and insulin aspart were spiked with a known amount of LPS, and these were used to perform the LPS detection. LPS-spiked sodium chloride and compound sodium lactate matrices (although being seemingly simple infusion solutions they are commercial products that might contain unknown additives, e.g., stabilizer that interferes with LPS) give similar fluorescence response (F/ F0) as in LPS-spiked PBS buffer (see Figure S4 in the Supporting Information), suggesting small/moderate matrix interferences. However, the insulin sample is more complicated, and insulin itself or additives in the sample solution inhibits the sensor response. While the inhibition mechanism is still unknown, the matrix effect by the insulin aspart solution can be reduced by sample dilution. For example, by diluting the sample (six times or more) yields essentially the same result as for LPS in PBS at the same concentration (see Figure S5 in the Supporting Information). Therefore, we believe this assay format is applicable for the detection of LPS in commonly used infusion solutions and insulin aspart, provided that proper validation and calibration are performed. LPS is known to exhibit a bacteria-strain-specific structural diversity and variation in biological endotoxic activity.45,46 However, most LPS biosensors are only validated by LPS from a single bacteria strain.12,13,15,44 Little is known about the applicability/variability of biosensor response induced by LPS of different bacteria strains. To test whether this LPS assay format is also capable to detect LPS from other Gram-negative bacterial strains, LPS-sensing experiments were performed by

Figure 4. Fluorescence intensity ratio (F/F0) of the peptide−GO sensor in PBS buffer solution upon addition of 2 μM LPS from different Gram-negative bacteria, as well as 2 μL of 50-fold diluted fresh E. coli (strain DH5α, OD600 = 0.7). F and F0 are fluorescence intensities at 572 nm with and without LPS, respectively. Results are the average of two experiments.

The results given above have several implications. First, it demonstrates that the sensor is capable of detecting LPS of a wide range of Gram-negative bacteria, which is critical for general application of endotoxin detection. The results also highlight the importance of considering LPS heterogeneity and response variability for validation and interpretation of the response from LPS biosensors. Moreover, it also offers an opportunity to design a strain-specific sensor to detect LPS from a desired bacterium, by tailored design of the peptide receptor. In many cases, the detection and identification of specific bacteria strain is required for clinicians to diagnose infectious diseases and to prescribe proper treatment. The importance of these results stems from the fact that, currently, there is no fast and reliable technique that allows a specific pathogen to be identified.47 We further extended our sensing work to detect living bacteria, motivated by the fact that LPS is the major component and located on the surface of Gramnegative bacterial outer membrane. Indeed, results in Figure 4 show that significant fluorescence increase (F/F0 = 2.4) can be induced immediately upon adding a 50-fold diluted fresh E. coli (strain DH5α, OD600 = 0.7). The high sensor response, even at low bacterial concentrations, offers great promise for rapid and sensitive detection of living bacteria in solutions, without employing tedious sample treatment and extracting procedures.

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CONCLUSIONS In summary, we have shown that a synthetic peptide−GO assembly can function as a robust fluorescence turn-on sensor for rapid, selective, and sensitive detection of LPS/endotoxin in aqueous solution, without the need of cumbersome sample preparation and storage at room temperature. The calculated detection limit of the sensor is 130 pM, which is the lowest among all synthetic LPS sensors reported so far. Moreover, this sensor is very specific for LPS, compared to other anionic D


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(16) He, Q.; Wu, S.; Yin, Z.; Zhang, H. Chem. Sci. 2012, 3, 1764− 1772. (17) Huang, X.; Qi, X.; Boey, F.; Zhang, H. Chem. Soc. Rev. 2012, 41, 666−686. (18) Xie, L.; Ling, X.; Fang, Y.; Zhang, J.; Liu, Z. J. Am. Chem. Soc. 2009, 131, 9890−9891. (19) Chen, P.; Yin, Z.; Huang, X.; Wu, S.; Liedberg, B.; Zhang, H. J. Phys. Chem. C 2011, 115, 24080−24084. (20) Peng, C.; Hu, W.; Zhou, Y.; Fan, C.; Huang, Q. Small 2010, 6, 1686−1692. (21) Loh, K. P.; Bao, Q.; Eda, G.; Chhowalla, M. Nat. Chem. 2010, 2, 1015−1024. (22) Liang, H.; Zhang, X.-B.; Lv, Y.; Gong, L.; Wang, R.; Zhu, X.; Yang, R.; Tan, W. Acc. Chem. Res. 2014, 47, 1891−1901. (23) Li, F.; Chao, J.; Li, Z.; Xing, S.; Su, S.; Li, X.; Song, S.; Zuo, X.; Fan, C.; Liu, B.; Huang, W.; Wang, L.; Wang, L. Anal. Chem. 2015, 87, 3877−3883. (24) Jang, H.; Kim, Y.-K.; Kwon, H.-M.; Yeo, W.-S.; Kim, D.-E.; Min, D.-H. Angew. Chem. 2010, 122, 5839−5843. (25) Dong, H.; Gao, W.; Yan, F.; Ji, H.; Ju, H. Anal. Chem. 2010, 82, 5511−5517. (26) He, S.; Song, B.; Li, D.; Zhu, C.; Qi, W.; Wen, Y.; Wang, L.; Song, S.; Fang, H.; Fan, C. Adv. Funct. Mater. 2010, 20, 453−459. (27) Gao, L.; Lian, C.; Zhou, Y.; Yan, L.; Li, Q.; Zhang, C.; Chen, L.; Chen, K. Biosens. Bioelectron. 2014, 60, 22−29. (28) Zhao, X.-H.; Kong, R.-M.; Zhang, X.-B.; Meng, H.-M.; Liu, W.N.; Tan, W.; Shen, G.-L.; Yu, R.-Q. Anal. Chem. 2011, 83, 5062−5066. (29) Huang, J.; Gao, X.; Jia, J.; Kim, J.-K.; Li, Z. Anal. Chem. 2014, 86, 3209−3215. (30) Tan, X.; Chen, T.; Xiong, X.; Mao, Y.; Zhu, G.; Yasun, E.; Li, C.; Zhu, Z.; Tan, W. Anal. Chem. 2012, 84, 8622−8627. (31) Liu, Z.; Chen, S.; Liu, B.; Wu, J.; Zhou, Y.; He, L.; Ding, J.; Liu, J. Anal. Chem. 2014, 86, 12229−12235. (32) Zhang, M.; Yin, B.-C.; Wang, X.-F.; Ye, B.-C. Chem. Commun. 2011, 47, 2399−2401. (33) Chang, H.; Tang, L.; Wang, Y.; Jiang, J.; Li, J. Anal. Chem. 2010, 82, 2341−2346. (34) Kwak, S.-Y.; Yang, J.-K.; Jeon, S.-J.; Kim, H.-I.; Yim, J.; Kang, H.; Kyeong, S.; Lee, Y.-S.; Kim, J.-H. Adv. Funct. Mater. 2014, 24, 5119− 5128. (35) Lu, C.-H.; Li, J.; Zhang, X.-L.; Zheng, A.-X.; Yang, H.-H.; Chen, X.; Chen, G.-N. Anal. Chem. 2011, 83, 7276−7282. (36) Wang, Z.; Huang, P.; Bhirde, A.; Jin, A.; Ma, Y.; Niu, G.; Neamati, N.; Chen, X. Chem. Commun. 2012, 48, 9768−9770. (37) Huang, H.; Feng, Z.; Li, Y.; Liu, Z.; Zhang, L.; Ma, Y.; Tong, J. Anal. Methods 2015, 7, 2928−2935. (38) Liu, J.; Liu, G.; Liu, W.; Wang, Y. Biosens. Bioelectron. 2015, 64, 300−305. (39) Zhang, H.; Huang, H.; Lin, Z.; Su, X. Anal. Bioanal. Chem. 2014, 406, 6925−6932. (40) Suzuki, M. M.; Matsumoto, M.; Yamamoto, A.; Ochiai, M.; Horiuchi, Y.; Niwa, M.; Omi, H.; Kobayashi, T.; Takagi, T. J. Microbiol. Methods 2010, 83, 153−155. (41) Li, D.; Muller, M. B.; Gilje, S.; Kaner, R. B.; Wallace, G. G. Nat. Nanotechnol. 2008, 3, 101−105. (42) Zhou, X.; Lu, G.; Qi, X.; Wu, S.; Li, H.; Boey, F.; Zhang, H. J. Phys. Chem. C 2009, 113, 19119−19122. (43) Kim, B.; Jung, I. H.; Kang, M.; Shim, H.-K.; Woo, H. Y. J. Am. Chem. Soc. 2012, 134, 3133−3138. (44) Sun, J.; Ge, J.; Liu, W.; Wang, X.; Fan, Z.; Zhao, W.; Zhang, H.; Wang, P.; Lee, S.-T. Nano Res. 2012, 5, 486−493. (45) Schromm, A. B.; Brandenburg, K.; Loppnow, H.; Moran, A. P.; Koch, M. H. J.; Rietschel, E. T. H.; Seydel, U. Eur. J. Biochem. 2000, 267, 2008−2013. (46) Lerouge, I.; Vanderleyden, J. FEMS Microbiol. Rev. 2002, 26, 17−47. (47) Fournier, P.-E.; Drancourt, M.; Colson, P.; Rolain, J.-M.; Scola, B. L.; Raoult, D. Nat. Rev. Microbiol. 2013, 11, 574−585.

biological interferences, and it also enables selective detection of LPS from different bacterial strains as well as LPS on the membrane of E. coli. Furthermore, the proposed assay can easily be read by simple and widely distributed benchtop spectrometers (e.g., plate readers). In the high concentration regime, the response could even be detected from the color by the naked eye. Undoubtedly, the fluorescence turn-on sensor offers great potential for endotoxin/LPS detection. In addition, the assay format could also be used for the detection of other biotoxins, biomarkers, and even pathogenic bacteria. Finally, compared to chemical attachment, physical adsorption of the dye-labeled receptor to GO enables release from the surface triggered by competitive binding. This binding triggered release strategy offers a general platform for many other target molecules (proteins, DNA, PNA, aptamers, etc.).

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ASSOCIATED CONTENT

* Supporting Information Downloaded by RUTGERS UNIV on September 1, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.analchem.5b02270

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02270. Details of the GO characterization by AFM and SEM, LPS sensing and control experiments, specificity test, and LPS detection in complex matrices (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Chia Wei Sheng for providing E. coli bacteria for bacteria sensing experiments. This work was supported by the School of Materials Science and Engineering and the Provost Office, Nanyang Technological University, Singapore.

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REFERENCES

(1) Beutler, B.; Rietschel, E. T. Nat. Rev. Immunol. 2003, 3, 169−176. (2) Qureshi, K.; Rajah, A. Br. J. Med. Pract. 2008, 1 (2), 7−12. (3) Munford, R. S. Annu. Rev. Pathol.: Mech. Dis. 2006, 1, 467−496. (4) Tidswell, M.; LaRosa, S. P. Expert Rev. Anti-Infect. Ther. 2011, 9, 507−520. (5) Beutler, B.; Poltorak, A. Crit. Care Med. 2001, 29, S2−S6. (6) Lourenço, F. R.; Botelho, T. D. S.; Pinto, T. D. J. A. PDA J. Pharm. Sci. Technol. 2012, 66, 542−546. (7) Morita, T.; Nakamura, T.; Miyata, T.; Iwanaga, S. Prog. Clin. Biol. Res. 1985, 189, 53−66. (8) Balagopal, A.; Gama, L.; Franco, V.; Russell, J. N.; Quinn, J.; Higgins, Y.; Smeaton, L. M.; Clements, J. E.; Thomas, D. L.; Gupta, A. PLoS One 2012, 7, e41258. (9) Elin, R. J.; Wolff, S. M. J. Infect. Dis. 1973, 128, 349−352. (10) Ding, J.; Ho, B. In Endotoxins: Structure, Function and Recognition; Wang, X., Quinn, P. J., Eds.; Springer: Dordrecht, The Netherlands, 2010; pp 187−208. (11) Rangin, M.; Basu, A. J. Am. Chem. Soc. 2004, 126, 5038−5039. (12) Voss, S.; Fischer, R.; Jung, G.; Wiesmüller, K.-H.; Brock, R. J. Am. Chem. Soc. 2007, 129, 554−561. (13) Wu, J.; Zawistowski, A.; Ehrmann, M.; Yi, T.; Schmuck, C. J. Am. Chem. Soc. 2011, 133, 9720−9723. (14) Sun, J.; Ge, J.; Liu, W.; Wang, X.; Fan, Z.; Zhao, W.; Zhang, H.; Wang, P.; Lee, S.-T. Nano Res. 2012, 5, 486−493. (15) Lan, M.; Wu, J.; Liu, W.; Zhang, W.; Ge, J.; Zhang, H.; Sun, J.; Zhao, W.; Wang, P. J. Am. Chem. Soc. 2012, 134, 6685−6694. E


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