Amplified Zinc Signal at a Nanocarbon Film Electrode for

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Cite This: ACS Appl. Nano Mater. 2018, 1, 5425−5429

Amplified Zinc Signal at a Nanocarbon Film Electrode for Lipopolysaccharide Detection Dai Kato,*,† Yoshio Suzuki,† Kyoko Yoshioka,† Tomoyuki Kamata,† Masami Todokoro,‡ and Osamu Niwa§ †

National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan JNC Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan § Saitama Institute of Technology, 1690 Fusaiji, Fukaya, Saitama 369-0293, Japan Downloaded via UNIV OF WINNIPEG on November 3, 2018 at 05:29:31 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: We report an electrochemical method for detecting low concentrations of lipopolysaccharide (LPS), based on amplified zinc (Zn) signals on a sputtered nanocarbon film electrode. The nanocarbon film electrode allowed us to detect Zn2+ ions at a lower concentration than a conventional glassy carbon electrode using anodic stripping voltammetry. This was due to the efficient accumulation and detection of Zn2+ ions with very low noise made possible by the ultraflat nanocarbon film surface. The amplified Zn signals from a newly synthesized Zn-complex-based probe were successfully used to detect LPS with low concentrations without employing conventional reagents. KEYWORDS: sputtered nanocarbon film, anodic stripping voltammetry, zinc ion, Zn-complex-based probe, lipopolysaccharide

S

negative potentials for preconcentration and oxidation at a working electrode. Electrode materials are therefore crucial in regards to realizing a high-performance platform for the electrochemical detection of biomolecules based on ASV measurements of Zn2+ ions. We recently reported an ultraflat nanocarbon film consisting of a nanocrystalline sp2/sp3 hybrid structure formed by electron cyclotron resonance sputtering15 or unbalanced magnetron sputtering.16 These film electrodes exhibited excellent electrochemical properties including a low background current and a wide potential window. These properties allowed us to detect various biomolecules by direct oxidation15−18 and heavy-metal ions (Cd2+ and Pb2+) by ASV.19 With Zn2+ determination by ASV, more negative applied potential is needed than for Cd2+and Pb2+ as described above.11,13,14,19 We expect the nanocarbon film electrode to be suitable for use in electrochemical Zn2+-ion detection because of its wide potential window and low noise current. Here we report an electrochemical method for detecting low concentrations of biomolecule LPS, based on Zn signals amplified at the nanocarbon film electrode. LPS, also known as endotoxin, is a major component of the outer membrane of Gram-negative bacteria. LPS must be removed from biological products that are administered

ignal amplification for chemical/biosensing is crucial in terms of realizing high levels of performance in regards to, for example, sensitivity and detection limit. With electrochemical methods, the enzymatic redox cycling1 and anodic stripping voltammetry (ASV) of heavy-metal ions2 are key signal amplification techniques. ASV is a powerful electroanalytical technique with which to determine trace heavy-metal ions with high sensitivity because it includes a step for preconcentrating heavy-metal ions on a solid electrode surface.2 This leads to the highly sensitive detection of target molecules as a result of the amplified metal-ion signal when constructing an electrochemical sensing system using metalbased labeling. Wang’s group has reported DNA detection using quantum dot (QD)-labeled oligonucleotides.3 Various kinds of metal-based labeling techniques have been reported ranging from inorganic QDs3 to metalloorganic complexes.4 Zinc (Zn)-based compounds are widely used for chemical and biochemical probes4−10 because Zn can be readily synthesized with optically active compounds and has relatively low toxicity. Thus, amplified Zn signals from Zn-based probes will enable us to detect trace levels of such biomolecules as lipopolysaccharide (LPS) and cytokine. ASV has been used to determine Zn2+ ions by employing mercury (Hg),3,11 bismuth (Bi),12 and boron-doped diamond (BDD)11,13,14 based electrodes, which can detect low concentrations of Zn2+ ions [typically on the order of 1−100 ppb (ng mL−1)]. These electrode materials commonly have a relatively wide potential window, especially in the cathodic direction, because Zn2+ ions exhibit relatively © 2018 American Chemical Society

Received: September 20, 2018 Accepted: October 17, 2018 Published: October 17, 2018 5425

DOI: 10.1021/acsanm.8b01663 ACS Appl. Nano Mater. 2018, 1, 5425−5429

Letter

ACS Applied Nano Materials

Figure 1. (a) Schematic illustration of a measurement using a combination consisting of an LPS-affinity microparticle, a Zn-complex-based probe for LPS detection (LPS probe), and an ultraflat nanocarbon film electrode: (1) An LPS sample was captured on LPS-affinity microparticles, and then (2) an LPS probe consisting of Zn-complex parts and a LPS-affinity peptide was captured on the LPS-adsorbed microparticles (accumulation of the LPS probe). The adsorbed LPS probe was treated with an acid solution, and finally (3) the Zn2+-ion concentration of the treated solution was measured by using our nanocarbon film electrode in a weak alkaline solution. (b) Chemical structure of the Zn-based LPS probe.

(LPS probe, Figure 1b), and an ultraflat nanocarbon film electrode. An LPS sample was captured on microparticles modified with poly(ε-lysine) (ε-PL) with a high affinity for LPS (Cellufine ET clean S, JNC Corp.), and then the LPS probe, consisting of Zn-complex parts and a LPS-affinity peptide, was captured on the LPS-adsorbed microparticles via the interaction between the LPS and the peptide with LPS affinity. The adsorbed LPS probe was treated with an acid solution, and finally we measured the Zn2+-ion concentration of the treated solution using our nanocarbon film electrode. The microparticles contribute to the accumulation of LPS and the subsequent LPS probe. Moreover, Zn2+ ions also accumulate on the nanocarbon film electrode thanks to the effective preconcentration step realized with the ASV technique. That is, the amplified Zn signal on the nanocarbon film electrode can be indicative of LPS even at a low concentration. To achieve this, we synthesized a new Zn-based LPS probe (Figure 1b) as described in Scheme S1. The Zn-based complex part, namely, zinc(II) dipicolylamine (Zn-DPA), is well-known in relation to the optical sensing of phosphate ions and proteins.5−10 One of the authors has already developed a ZnDPA-based reagent for fluorescent gel staining to detect proteins.6 In regards to the LPS-affinity part, we selected an LPS binding peptide (Kd = 10−11 M) obtained using the phage display method developed by Suzuki and co-workers.29,30 In regards to the working electrode for detecting Zn2+ ions released from the LPS probe, we used a nanocarbon film electrode with an sp2/(sp2 + sp3) ratio of 0.5. This electrode has a sufficiently wide potential window with a low background current resulting from its ultraflat surface [average surface roughness (Ra) = 0.1−0.2 nm].16

intravenously (e.g., infusion fluids and injections including water precursor) because LPS triggers a wide variety of biological effects such as sepsis and septic shock in humans,20,21 even at low concentrations (ng mL−1). Indeed, these effects are responsible for 250000 deaths annually in the United States.22 Therefore, this makes it essential to measure the LPS content to ensure the quality of these cellular products. The Limulus amoebocyte lysate (LAL)-based assay technique is now the gold standard in regards to a conventional LPS assay thanks to its extremely high sensitivity (or low detection limit) and selectivity against LPS.20,21 However, it is relatively expensive and time-consuming, especially when used in the lower LPS concentration region. Therefore, LAL reagent-free LPS analyses, including fluorometric, colorimetric, and chemiluminescent methods, have recently been developed.23−28 Lan and co-workers reported a fluorometric LPS sensor realized by using copolythiophene derivatives,23 which exhibited a low detection limit (2.7 ng mL−1) with a very rapid (several minutes) assay. On the other hand, Xu and co-workers reported a colorimetric aptasensor for LPS that exhibited superior performance including the provision of a low detection limit (50 pg mL−1) within 4 h.27 However, it is difficult to obtain both good sensitivity and selectivity while maintaining rapid analysis. Indeed, recent reports on LPS sensors tend to involve relatively complicated and time-consuming procedures. Therefore, the LAL-free LPS detection systems still require improved sensitivity and selectivity with simple materials and procedures. Figure 1a outlines the concept behind our LPS detection methodology, which realizes a superior performance without an LAL reagent by using the combination of LPS-affinity microparticles, a Zn-complex-based probe for LPS detection 5426

DOI: 10.1021/acsanm.8b01663 ACS Appl. Nano Mater. 2018, 1, 5425−5429

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ACS Applied Nano Materials

surface.16 This ultraflat surface also provided a saturated signal at a higher Zn concentration under optimized conditions. Proof-of-concept experiments for LPS detection according to Figure 1a were performed with the synthesized LPS probe. Here, a flow measurement system was constructed to perform the LPS measurement, as given in Figure S3 and Table S1. Figure 3a shows the ASV results obtained with and without an

We estimated the electrochemical properties of the nanocarbon film electrode in relation to Zn2+ detection. We also used a commercially available glassy carbon (GC) electrode to measure Zn2+ ions as a comparison. Figure 2a shows typical

Figure 3. Electrochemical detection of LPS by the LPS probe: (a) LPS concentration dependence of voltammograms and (b) the obtained calibration at the nanocarbon film electrode. The SWV conditions are the same as those in Figure 2.

LPS sample. With the LPS samples, we obtained clear ASV peaks that depended on the LPS concentration with relatively wide concentration ranges. This result clearly indicates that the Zn signal works well as an indicator of the LPS concentration in spite of three points upon calibration at the present stage. From the calibration curve (r = 0.984 in Figure 3b), the detection limit was 200 pg mL−1 even without LAL reagents. Several researchers have developed the LAL reagent-free LPS analysis method.23−28 Compared with these methods, our proposed method revealed rapid assay even with the simpler procedure. Indeed, our method comprising four steps requires 2.5 h (excluding the washing processes), whereas other methods have more than four steps that require long measurement periods (3.5−7.5 h, excluding the washing processes). The obtained current value (400 nA) from LPS (2 ng mL−1) was estimated to be about 22.9 nmol of Zn2+ (11.45 nmol of the LPS probe; the calculation method is shown in the Supporting Information, SI), showing that the LPS probe exhibited a high LPS binding capacity (163 ng of LPS/mg of peptide), which was equivalent to the previously reported value (170 ng of LPS/mg of peptide).29,30 This slight reduction would be predominantly due to the steric hindrance of the Zn-complex parts. Nevertheless, these results indicate that the peptide introduced into our probe provides results as good as those of previous reports. In fact, the obtained binding capacity value was superior to that of polymyxin B (80 ng of LPS/mg), which is well-known as an LPS binding molecule with high affinity.30 In regards to the reproducibility, when we measured Zn2+ ions (50 ng mL−1) by using the nanocarbon film, the RSD value (n = 3) was 3.1%, whereas our LPS sensor exhibited an RSD value of 6.6% (n = 3) for 20 ng mL−1 LPS despite the fact that both exhibited the same currents (same Zn concentrations). Taking account of the fact that our sensor system consists of an LPS reaction part (minicolumn) and a detection part (nanocarbon film electrode), this RSD decrease reflects the low reproducibility of the LPS reaction measurement. We conducted a selectivity test using β-D-glucan, which is known to be an interference molecule for LPS determination

Figure 2. Typical SWV curves of Zn2+ at the nanocarbon film (a) and GC (b) electrodes, measured in a 0.1 M, pH 8.0, ammonium buffer. The preconcentration time is 240 s. The preconcentration potential is −1.5 V. Amplitude = 25 mV, ΔE = 2 mV, f = 40 Hz. (c) Calibration curves of Zn2+ at the nanocarbon film (red) and GC (blue) electrodes. (d) Enlargement of Figure 2c.

square-wave voltammetry (SWV) curves of Zn2+ ions obtained by ASV using our nanocarbon film electrode, measured under optimized conditions, as summarized in Figures S1 and S2. We observed clear oxidation peak currents for the preconcentrated Zn0 on the surface around −1.1 V versus Ag/AgCl. The GC electrode exhibited similar results (Figure 2b). In both cases, a gradual shift of the peak potential was observed with increasing Zn concentration because of the change in the transition of the stripping surface from carbon to Zn with increasing Zn concentration.19,31 The lowest concentrations actually observed in the voltammograms were 0.5 ng mL−1 on the nanocarbon film electrode. From calibration curves in linear ranges (r = 0.981 in the region of 0.5−250 ng mL−1; Figure 2d), the detection limit of the Zn2+ ions at the nanocarbon film electrode was 1.0 ng mL−1 (S/N = 3), which was low compared with that at the GC electrode (4.8 ng mL−1). The obtained result was the same as, or slightly better than, those obtained with recently developed Hg, Bi, and BDD electrodes.11,13,14 When we take account of the full width at halfmaximum (fwhm) of the ASV peak (100 ng mL−1 Zn2+), the nanocarbon film exhibited an fwhm of 64 mV compared with 70 mV at GC, which is comparable to the value reported by Swain (60 mV for BDD in the presence of the same Zn2+ concentration)11 in spite of the different measurement conditions. It is noteworthy that this performance was achieved using a pure carbon-based film electrode with no need for a Bi- or Hg-based electrode. This was because the nanocarbon film electrode exhibited a low background current compared with the GC electrode, thanks to its ultraflat 5427

DOI: 10.1021/acsanm.8b01663 ACS Appl. Nano Mater. 2018, 1, 5425−5429

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by the LAL method.20 In this system, we observed no current response caused by the reaction of Zn2+ ions with β-D-glucan (100 ng mL−1; Figure 4a) even though its concentration was

Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Dai Kato: 0000-0003-0787-0562 Yoshio Suzuki: 0000-0002-7305-1242 Kyoko Yoshioka: 0000-0003-3332-0038 Osamu Niwa: 0000-0003-0676-6827 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (Grant 25288071 to D.K.) from the Ministry of Education, Culture, Science, Sports and Technology of Japan. We thank Masumi Hirashima (formerly at AIST) and Shuji Sasaki (formerly at JNC Corp.) for their fine contributions to this work.

Figure 4. (a) Electrochemical response of β-D-glucan (red line) obtained with the LPS probe. The black line represents the background scan. The SWV conditions are the same as those in Figure 2. (b) Correlation between our results and those obtained for LPS samples with the conventional LAL method.

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ABBREVIATIONS ASV = anodic stripping voltammetry BDD = boron-doped diamond Bi = bismuth DPA = dipicolylamine ε-PL = poly(ε-lysine) fwhm = full width at half-maximum GC = glassy carbon Hg = mercury LAL = Limulus amoebocyte lysate LPS = lipopolysaccharide QD = quantum dot SWV = square-wave voltammetry Zn = zinc

much higher than that of the LPS that we measured. This was because the ε-PL group used to modify the microparticle surface did not interact with the β-D-glucan molecule. The selectivities for glucose, peptidoglycan, and lipoteichoic acid were also estimated in the same way as that for β-D-glucan, and very small responses were obtained, as shown in Figure S4. Moreover, it is noteworthy that the ε-PL-group-modified microparticle exhibited the highly selective adsorption of LPS, whereas no adsorption of not only the β-D-glucan but also various proteins such as albumin, myoglobin, lysozyme, and so on.32 Moreover, we confirmed the accuracy of our results by a comparison with those obtained with the conventional LAL method (see the SI; with a range of 0.2−20 ng mL−1 LPS), as shown in Figure 4b. The results correlated well with a correlation coefficient of 0.999 (p < 0.03). Therefore, we attributed the Zn signal obtained in this study to the affinity of LPS. In conclusion, we successfully developed an electrochemical LPS detection system based on amplified Zn signals on a nanocarbon film electrode, which did not require Hg and Bi electrodes or conventional LAL reagents. Much higher sensitivity is required when using water injection because the LPS concentration limit is imposed by international regulations (e.g., around several tens of picograms per milliliters33). Therefore, we must undertake a further investigation to realize a quantitatively superior performance without the need for conventional LAL reagents. Anyway, our approach is highly advantageous for detecting both the target biomolecules from a Zn-based probe and Zn2+ ions with a lower concentration than a GC electrode. This sensing platform can also be applied to various biomolecules with trace-level concentrations by changing the recognition part of the developed probe.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b01663. Full experimental details, Scheme S1, Table S1, and Figures S1−S4 (PDF) 5428

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DOI: 10.1021/acsanm.8b01663 ACS Appl. Nano Mater. 2018, 1, 5425−5429