Hierarchical CNTs@CuMn Layered Double Hydroxide Nanohybrid

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Hierarchical CNTs@CuMn Layered Double Hydroxide Nanohybrid with Enhanced Electrochemical Performance in H2S Detection from Live Cells Muhammad Asif, Ayesha Aziz, Zhengyun Wang, Ghazala Ashraf, Junlei Wang, Hanbo Luo, Xuedong Chen, Fei Xiao, and Hongfang Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04685 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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Analytical Chemistry

Hierarchical CNTs@CuMn Layered Double Hydroxide Nanohybrid with Enhanced Electrochemical Performance in H2S Detection from Live Cells Muhammad Asif,†,‡ Ayesha Aziz,† Zhengyun Wang,† Ghazala Ashraf,† Junlei Wang,† Hanbo Luo,†† Xuedong Chen,‡ Fei Xiao,*,† and Hongfang Liu*,† † Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, Hubei Key Laboratory of Material Chemistry and Service Failure, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, P. R. China ‡ State Key Laboratory of Digital Manufacturing Equipment and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China †† No.1 Middles School Affiliated to Central China Normal University, Wuhan, 430070, P. R. China

*Corresponding Authors: [email protected] (F. Xiao) [email protected] (H. Liu)

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ABSTRACT: The precise monitoring of H2S has aroused immense research interests in biological and biomedical fields since it is exposed as third endogenous gasotransmitter. Hence, there is an urgent requisite to explore ultrasensitive and economical H2S detection system. Herein, we report a simple strategy to configure extremely sensitive electrochemical sensor with 2D nanosheetshaped layered double hydroxide (LDH) wrapped carbon nanotubes (CNTs) nanohybrid (CNTs@LDH), where a series of CNTs@CuMn-LDH nanohybrids with varied amount of LDH nanosheets grafted on conductive CNTs backbone has been synthesized via facile coprecipitation approach. Taking the advantage of the unique core-shell structure, the integrated electrochemically active CuMn-LDH nanosheets on conductive CNTs scaffold, the maximum interfacial collaboration, and the superior specific surface area with plethora of surface active sites and ultrathin LDH layers, the as-prepared CNTs@CuMn-LDH nanoarchitectures have exhibited superb electrocatalytic activity towards H2S oxidation. Under the optimum conditions, the electrochemical sensor based on CNTs@CuMn-LDH nanohybrid shows remarkable sensing performances for H2S determination in terms of wide linear range and low detection limit of 0.3 nM (S/N=3), high selectivity, reproducibility and durability. With marvelous efficiency achieved, the proposed sensing platform has been practically used in in-situ detection of abiotic H2S efflux produced by sulfate reducing bacteria and real-time in-vitro tracking of H2S concentrations from live cells after being excreted by stimulator which in turn might serve as early diseases diagnosis. Thus, our core-shell hybrid nanoarchitectures fabricated via structural integration strategy will open new horizon in material synthesis, biosensing system and clinical chemistry. KEYWORDS: Core-shell structure, CuMn layered double hydroxide, Electrochemical biosensor, H2S determination, SRB supernatant, Living cells

INTRODUCTION In modern era, hydrogen sulfide (H2S) with diverse physiological profile has unveiled a significant signaling role in biology and biomedicine for precise monitoring of chronic diseases, since it has been recognized as third endogenous gasotransmitter after carbon monoxide and nitric oxide.1,2 In living organisms, it is engendered by cysteine biosynthesis pathway through 2 ACS Paragon Plus Environment

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several enzymatic reactions and its ordinary concentrations regulate various pathological functions such as antioxidation, neurotransmission, cell proliferation and vascular inflammation.3 In particular, H2S produced in cardiovascular system of mammals is found to lead to dilation of blood vessels and lowering of blood pressure. The possible cytoprotective ability of H2S during different models of cellular injury is somewhat associated with its aptitude of neutralizing reactive oxygen species.4 Nevertheless, gigantic accumulation of H2S in cellular environment is extremely detrimental to living systems and can be associated with numerous enduring diseases, such as alzheimer's disease, down's syndrome, fatal kidney disease, diabetes and cancer.3,5 The monitoring of endogenous sulfide efflux in biological fluids is highly challenging because of its short lifetime and has great impact towards the guidance of clinical investigation.6 Encouraged by physiopathological roles of abiotic H2S, its real-time precise cellular detection is extremely mandatory to avoid lethal attacks and further offering reliable diagnosis of neurological syndromes. Up to now, several analytical approaches towards H2S measurement have been reported such

as

spectrophotometry,7

fluorimetry,8

chemiluminescence,

chromatography

and

electrochemistry.9 Chemiluminescence is often avoided to be used in biological samples because of its high working temperature, delayed response and recovery time. Fluorescence, instead of being able to image cell-based sulfides in live cells, is still unable to track in-situ fluctuations of H2S in real-time10. Especially, electrochemical transducer based biosensors have conquered wealth of interests of electrochemists due to their excellent performance, portability and realtime in-vitro as well as in-vivo monitoring capabilities.11 Moreover, commonly used biosensors for H2S detection are inhibitive and microbial biosensors, where the former method is based on inhibition effect of enzyme activity upon H2S exposure.12,13 However, inhibitive enzymes suffer instability, complicated immobilization, high cost, scarcity and poor anti-interferrence ability against cyanide ions.14 In case of microbial biosensors, microbes are used as biological recognition component that can directly oxidize H2S molecules, but still possess certain drawbacks including complex cultivation procedure for microbes and slow H2S oxidation rate.15 Regrettably, electrochemical oxidation of H2S normally takes place at higher overpotential which may result in interferences caused by electroactive species existing in central nervous system. Though, some special metal electrodes including Ni and V2O5 can be used at low potential for electrochemical oxidation of H2S but electrochemical reactions need to be operated at strong 3 ACS Paragon Plus Environment


acidic or basic conditions which may excrete H2S from bound sulfane sulfur and causes overestimation.16 Therefore, exploration into nanomaterial based nonenzymatic biosensors with superb performances in terms of good sensitivity and anti-interferrence ability in H2S monitoring has attained always undoubted priority in sensing systems.17 Inspired by distinct features and intrinsic electrocatalytic abilities of layered double hydroxide (LDH) materials, they have been widely employed in cancer therapy, catalysis and electrochemical sensors.18 Regardless of great development made in LDH materials, they still suffer from low conductivity that confines the efficiency of redox process.19 To circumvent this conundrum, incorporation of LDH materials with conductive carbon substrate such as carbon nanotubes (CNTs) has been expected as an ideal approach to accomplish overall optimized electrochemical performance.20,21 Moreover, CNTs being high electrically conductive backbone, robust network with large surface area and capability to be functionalized may facilitate as electron collection/transportation during catalytic reactions enabling fast redox processes.22 Motivated by this necessity, we report hierachical hybrid nanoarchitectures by structurally integrating high quality CuMn-LDH nanoplates on functionalized CNTs networks (CNTs@CuMn-LDH) and explore their electrocatalytic behavior towards H2S oxidation. So far as we know, it has never been reported previously. The biosensing system employed in tracking H2S flux from live cells is necessary to be operated at physiological conditions. Ideally, the most electrochemically detectable form is HS− ions which exist around 80% in buffer solution at physiological pH. Interestingly, the electrode modified with CNTs@CuMn-LDH nanohybrids performs well at biological pH (7.4) which is necessary to provide the accurate pH environment for sufficient transformation of H2S to the electrochemically detectable HS− form. In this work, different CNTs@CuMn-LDH hierarchical nanocomposites with varied amount of loaded LDH nanosheets on CNTs framework have been fabricated by facile co-precipitation method, which is simple, economical and holds maximum loading capacity. The electrode modified with CNTs@CuMn-LDH exhibits superb catalytic aptitude for H2S oxidation owing to nanoscale interfacial collaborations, where CuMn-LDH nanosheets provide enlarged surface area which ensures abundant redox reactions and CNTs backbone offers conductive pathways to facilitate fast electron shuttling. Benefitted from enhanced electrochemical sensing performances for H2S detection in terms of wide linear range and low detection limit of 0.3 nM, the as synthesized 4 ACS Paragon Plus Environment

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CNTs@CuMn-LDH have been practically applied in in vitro detection of SRB through the sensing of endogenous sulfide generated by characteristic metabolic process. In addition, proposed electrochemical biosensing system has also been employed in detection of cellular H2S excreted from human live cells.

EXPERIMENTAL SECTION Functionalization of CNTs. Typically, 500 mg CNTs were refluxed in concentrated 100 mL H2SO4 (98 wt%) and 35 mL H2O, 35 mL HNO3 (65 wt%) at 120 °C for 5 h. Finally, after centrifugation of mixture, washing with DI water was continuously carried out till neutral pH of product and then dried at 80 °C for further use.

Figure 1. Schematic illustration of various steps involved in the synthesis of hierarchical CNTs@CuMn-LDH nanohybrids and analytical performance for H2S detection. Synthesis of CNTs@CuMn-LDH Nanocomposites. The CNTs@CuMn-LDH hierachical nanocomposites with varied loading of LDH nanoplates on CNTs framework were fabricated by facile co-precipitation method, as illustrated in Figure 1. In a typical procedure, 20 mg of modified CNTs were dispersed in 100 mL DI water and kept under sonication for 30 min. Secondly, 50 mL of mixed solution containing Cu(NO3)2·3H2O (0.05 mM) and MnCl2·4H2O (0.025 mM) was added dropwise to the above CNTs dispersion under vigorous stirring. The pH of solution was maintained at 10±0.03 with 0.4 M NaOH solution throughout the experiment. A specific amount of H2O2 (25 µL) was injected to above suspension for oxidation of Mn2+ species. Then this colloidal solution was aged at 60 °C for 12 h. The centrifugation was done to wash the product with DI water several times and finally dried over night at ambient temperature. The 5 ACS Paragon Plus Environment


analogous reactions in the absence and presence of varied amounts of CNTs were carried out by workup similar to above mentioned procedure to fabricate control materials Table S1.

RESULTS AND DISCUSSION Material Characterization. The morphology and analytical performances of CNTs@CuMnLDH2 hybrid, denoted as CNTs@CuMn-LDH (where 20 mg of CNTs have been used as core, Table S1) have been evaluated. The ratio of metallic salts and CNTs are adjusted to develop hierarchical CNTs@CuMn-LDH core-shell morphology with intermediate thickness of CuMn LDH shell. The structural morphologies of CNTs@CuMn-LDH hybrid have been shown in Figure 2. A uniform coating of CuMn-LDH sheets has been achieved on the surface of CNTs through a facile in-situ growth method representing completely covered CNTs skeleton having open and porous structure as depicted by SEM image (Figure 2A). The resultant CNTs@CuMnLDH hybrid material is composed of numerous intertwined gauze-like nanosheets of LDH ensuring compact contact between CuMn-LDH and CNTs as shown in Figure 2B. It is further expected that a superior electron transportation and ion diffusion not only on electrode surface but also within the framework of hybrid nanoarchitectures would be attained by as-fabricated core-shell CNTs@CuMn-LDH composite.

Figure 2. (A & B) SEM images and (C) TEM image of CNTs@CuMn-LDH nanohybrids at different magnifications, (D) HRTEM image of CNTs@CuMn-LDH nanohybrids with lattice fringes of 0.25 nm, 0.27 nm and 0.34 nm Moreover, TEM characterization has been performed to yield better view on structural morphologies of hybrid core-shell nanoarchitectures. Figure S2 (A) presents hierachical core6 ACS Paragon Plus Environment

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shell structure assembled from nanosheet-like numerous units. The magnified images in Figure 2C and Figure S2 (B) illustrate that the resultant CNTs@CuMn-LDH hybrids hold almost transparent feature, representing its ultrathin property. Further it is concluded that petals of LDH are oriented almost horizontally along with few vertically onto the surface of CNTs network. Additionally, the CuMn-LDH nanoplates are strongly associated with each other and the layered graphitic walls of CNTs skeleton which can also be visualized because of ultrathin grafting of CuMn-LDH layers. The entire core of CNTs skeleton seems to be buried completely in the LDH layers at lower amount of CNTs than transitional ratio (Figure S1). The HRTEM image (Figure 2D) demonstrates the lattice fringes with d-spacing of 0.27 nm, 0.25 nm and 0.34 nm corresponding to the presence of Mn(III), Cu(II) and CNTs respectively.23 The lamellar fringe with interpalanar distance of 0.77 nm indicates interlayer spaces typically measured for (003) LDH plane (Figure S2C). The crystalline structures of hybrid and other control materials were investigated by typical XRD measurements and corresponding results have been presented in Figure 3A. The XRD pattern of CNTs consists of one peak at 26.3º demonstrating (002) crystal reflection of graphite layers. Regarding the XRD patterns of pristine CuMn-LDH and CNTs@CuMn-LDH hybrid, crystal planes of (003), (006), (009) (015), (018), (110), and (113) at 2θ value of 11.23°, 22.7° 34.5°, 39.3°, 47.1°, 60.3° and 61.7° respectively are attributed to hexagonal geometry of CuMn-LDH (JCPDS no. 22-0452), authenticating the phase transformation from precursor salts to CNTs@CuMn-LDH hybrid.24 The interlayer spacing of 0.77 nm is measured for (003) diffraction peak of LDH which is probably due to the intercalation of carbonate ions and water molecules in between the interlayer galleries. It is noteworthy that (002) crystal peak in CNTs@CuMn-LDH hybrid is noticeably decreased which undoubtedly indicates the successful grafting of CuMn-LDH on the surface of CNTs skeleton. Further by decreasing the amount of CNTs, the (002) diffraction peak is vanished in between the dozens of agglomerated LDH layers (Figure S3). These results indicate that CuMn-LDH has been thoroughly coated on the surface of CNTs backbone. The valence electronic configuration and element composition of composite were evaluated by employing XPS characterization. The wide scan spectrum of CNTs@CuMnLDH hybrid has been displayed in Figure 3B which indicates the distinct peaks assigned to Cu 2p, Mn 2p and Mn 3p along with two dominant signals of oxygen and carbon corresponding to charge compensating hydroxyl and carbonate ions present in LDH phase. 7 ACS Paragon Plus Environment


Figure 3. (A) The XRD pattern of pristine CNTs, CuMn-LDH and CNTs@CuMn-LDH, (B) Wide scan XPS survey spectra, (C) Cu 2p core level XPS spectra, and (D) Mn 2p core fitting XPS spectra of CNTs@CuMn-LDH sample Figure 3C represents the core-level spectrum of Cu 2p, which is further splitted into two peaks of Cu 2p3/2 and Cu 2p1/2 originated at 935.2 eV and 955.3 eV respectively, corresponding to Cu(II) phase. The appearance of two satellite peaks at around 943.4 eV and 963.2 eV is the fingerprint of existence of CuO species with d9 electronic configuration.25 The deconvoluted spectrum of Mn 2p has been displayed in Figure 3D with further splition into couple of peaks Mn 2p3/2 and Mn 2p1/2 at 642.4 eV and 654.05 eV having spin energy separation of 11.7 eV, which collectively are accredited to the presence of Mn(III) species.26,27 The deconvolution is carried out on Mn 2p spectrum aiming to confirm the characteristics peak of Mn3+ phase. Electrochemical Performance of CNTs@CuMn-LDH/GCE. To get insight into the effects of CNTs, the kinetics of catalytic processes on GCE modified with CNTs, CuMn-LDH and CNTs@CuMn-LDH hybrid were evaluated with electron impedance spectroscopy (EIS) using [Fe(CN)6]3‑/4‑ redox probe as presented by Figure 4A, which offers significant information about electron transfer parameters and bulk electrolyte properties. And the inset is the equipped Randle equivalence circuit specifying Rs, Rct, Cdl and W, as resistance of solution, charge transfer resistance, double layer capacitance, and Warburg constant, respectively. The results demonstrate that the CNTs@CuMn-LDH hybrid material possesses small electron transfer resistance of 541.1 Ω compared with pristine CuMn-LDH (936.9 Ω) and bare electrode (2427 Ω), suggesting its superb charge transport capabilities during electrochemical redox processes. 8 ACS Paragon Plus Environment

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The CNTs modified GCE exhibits a small Rct value of 316.3 Ω because of its good conductivity. The CuMn-LDH/GCE shows less Rct value than bare GCE, indicating fractional charge transfer ability of LDHs owing to the presence of holes within the layers of LDHs materials. The superb charge transfer properties of CNTs@CuMn-LDH hybrid might have arisen due to the synergistic effects of ultrathin CuMn-LDH nanoflakes, highly conductive CNTs framework and enlarged specific surface area, resulting in faster reaction kinetics. The ultrathin 2D nanosheets of CuMnLDH further ensure the intimate contact between electrocatalysts and electrode surface, amazing interfacial contact with electrolyte and target analyte and reduced ion-diffusion shuttling, hence increasing charge transportation. The typical cyclic voltammetric (CV) measurements were employed to investigate electrocatalytic abilities of electrode modified with CNTs@CuMn-LDH hybrid. Figure 4B represents the CV curves of CNTs@CuMn-LDH/GCE in 0.1 M PBS (pH 7.4) in the absence (black line) and presence (red line) of 0.5 mM H2S. In presence of blank PBS solution, a couple of minor peaks accredited to redox of Cu(II) to Cu(III) phases can be witnessed. While in the presence of 0.5 mM H2S, CNTs@CuMn-LDH/GCE exhibits a distinct oxidation peak at potential of 0.24 V. By increasing H2S concentration, corresponding oxidation peak current values are also enhanced linearly as demonstrated by DPV curves in Figure 4C and their relevant linear relationship has been displayed in Figure S4.

Figure 4. (A) EIS plots of bare GCE, CuMn-LDH/GCE, CNTs@CuMn-LDH/GCE and CNTs/GCE in 0.1 M KCl consisting of 1.0 mM K3Fe(CN)6 and 1.0 mM K4Fe(CN)6, Frequency range: 0.1-105 Hz. Inset is the equivalent circuit, (B) CV curves of CNTs@CuMn-LDH/GCE in 9 ACS Paragon Plus Environment


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0.1 M PBS in the absence (black line) and presence (red line) of 0.5 mM H2S, (C) CV responses of CNTs@CuMn-LDH/GCE in 0.1 M PBS with different concentrations of H2S. (D) CV curves of different modified electrodes in 0.5 mM H2S in 0.1 M PBS solution The possible catalytic mechanism of as-fabricated CNTs@CuMn-LDH hybrid towards oxidation of H2S can be predicted as follows; CuO + OH− 2CuOOH + H2S Mn2O3 + 2OH− 2MnO2 + H2S

CuOOH + e− 2Cu(OH)2 + S0 2MnO2 + H2O +2e− Mn2O3 + H2O + S0

(1) (2) (3) (4)

It is well-documented that Cu(III) and Mn(IV) species act as electron transfer mediators rather than their other oxide forms. It is also revealed that oxides and hydroxides are accountable to electro-oxidize H2S in an alkaline medium. During electrochemical reactions, Cu(II) and Mn(III) would be converted into Cu(III) and Mn(IV) respectively which consequently catalyze H2S to sulfur.28,29 The mixed oxides can improve catalytic activity towards H2S oxidation in alkaline medium which facilitates the transformation of Cu(III)/Cu(II) and Mn(IV)/Mn(III) redox couples. Additionally, a special feature associated with hydrotalcite like materials is the replacement of divalent cations by some trivalent cations, which creates positive holes within the LDH layers that are balanced by the presence of counteranions such as OH¯, CO32−, Cl−, NO3− located in the interlamellar region. These positive holes then act as charge carriers and enhance the electron transportation during the redox process. The electrocatalytic performances of CNTs@CuMn-LDH without and with different amount of CNTs have also been assessed as depicted by Figure 4D. The CNTs/GCE did not show any obvious current peak towards H2S oxidation. Among the hybrid nanomaterials, CNTs@CuMn-LDH2 exhibited striking catalytic activities towards oxidation of H2S in comparison with CNTs@CuMn-LDH1 and CNTs@CuMn-LDH3 having less or higher CNTs ratio. At low amount of CNTs, decreased oxidation current peak is due to the fact that CNTs seem to be completely buried in LDH layers which endow poor conductivity to the hybrid material, while at higher CNTs ratio, the surface of CNTs frameworks is not thoroughly covered by LDH nanoflakes, ultimately there is no perfectly 10 ACS Paragon Plus Environment

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core-shell structure offering excessive nanoscale interfaces for redox processes. The splendid electrochemical efficiencies of CNTs@CuMn-LDH hybrid with intermediate CNTs may come from synergistic contribution of following factors; i) functional integration of highly conducting CNTs and electrochemically active CuMn-LDH nanosheets, ii) well-interconnected conductive backbone of CNTs as core and compact shell of CuMn-LDH provide maximum interfacial collaboration to enhance overall electrocatalytic activity, iii) superior specific surface area, which in turn bestows the catalyst with plethora of surface active sites,19 iv) ultrathin layers of LDH guarantee the intimate interaction with substrate electrode, consequently upsurge interfacial contact area with electrolyte and reduce ion-diffusion distances of nanoscale dimensions. Furthermore, CV curves were also performed to investigate the effect of various pH of 0.1 M PBS as supporting electrolyte in the presence of 0.5 mM H2S at CNTs@CuMn-LDH/GCE (Figure S5). The pH range of 4.4 to 9.4 shows dynamic impact on the electrocatalytic oxidation ability of CNTs@CuMn-LDH in H2S determination. The enhancement in oxidation peak current is observed from pH 4.4 to 7.4 and the highest current is noticed at pH 7.4. While using PBS with pH higher than 7.4, gradual decline in peak current values for H2S oxidation is observed. So, pH 7.4 is selected as optimum pH for H2S sensing. Hydrogen sulfide is a week acid with pKa1 and pKa2 values as given below in following equations. H2S

⇌ HS−

pKa1 = 6.9

(5)

HS−

⇌

S2-

pKa2 = 14.15

(6)

At pH 7.4, 20% H2S, 80% HS− and 0% S2− exist in phosphate buffer while at extreme acidic and basic pH, H2S and S2- are the prominent forms respectively.30 Interestingly, the most electrochemically detectable form is HS− ion. The oxidation rate of H2S is slow at strong acidic and basic owing to the unavailability of most detectable HS− form. Consequently, slightly basic PBS with biological pH 7.4 is used and this pH is necessary to provide the factual pH environment for ample transformation of H2S to the electrochemically detectable HS− form. Further to employ the sensing platform in biological environment, PBS under physiological conditions has been used because the extreme acidic or basic conditions may cause overestimation of free hydrogen sulfide in real tissues.31 The sensitivity of as-synthesized CNTs@CuMn-LDH hybrid nanostructures was also examined with amperometric measurements towards H2S oxidation at a fixed potential of 0.24 11 ACS Paragon Plus Environment


V. Figure 5A portrays steady-state current-time (i.t) responses of CNTs@CuMn-LDH/GCE upon consecutive addition of H2S concentrations in stirring PBS (pH=7.4). The real-time detection limit with (S/N=3) has also been measured by CNTs@CuMn-LDH/GCE, which is as low as 0.3 nM (inset of Figure 5A). The magnified current-response curve has been placed in Figure 5B. A well-separated and rapid stepwise enhancement in the current signals has been achieved by spiking different aliquots of H2S, which swiftly reaches steady-state current feedback of ∼95% within 3 s, with wide linear sensing range from 8 nM to 2.9 mM (R2 =0.995). The rapidly and linearly increased current responses of CNTs@CuMn-LDH/GCE might be attributed to the effective diffusion and activation of H2S molecules on large surface active sites harvested by exceptionally rough surface of CuMn-LDH on CNTs framework and improved charge transport kinetics as well.

Figure 5. (A) Typical amperometric signals recorded with CNTs@CuMn-LDH/GCE upon successive additions of different H2S concentrations in 0.1 M stirring PBS at an applied potential of 0.24 V, inset is the amperometric response for real detection limit. (B) The magnified portion of i.t curve to several micromolar levels, (C) The linear calibration curve of current vs. H2S concentration, (D) Amperometric current records of electroactive interfering species upon 0.5 mM addition of each spike of H2S. The broad linear response curve between increasing concentrations of H2S and corresponding oxidation current is shown in Figure 5C. Our proposed biosensor outperforms compared with many other electrode materials used for the detection of H2S over the last few years as presented by Table S2, which reveals its brilliant performance in H2S sensing for practical presentation. Moreover, anti-interference capability of CNTs@CuMn-LDH/GCE was explored by employing 12 ACS Paragon Plus Environment

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amperometric measurements. Upon the addition of 5 mM of interfering species such as nitrates, chlorides, sulfates, thiosulfates, carbonates, cyanides and nitrites in 0.1 M stirring PBS at an applied potential of 0.24 V, no any obvious interference is observed. While amperometric current responses for 0.5 mM of H2S in the same PBS solution are significant, substantiating the superior anti-interferrence ability of CNTs@CuMn-LDH/GCE based biosensing system for detecting H2S effluxes (Figure 5D). In vitro Detection of H2S in Biological Samples. It is well-documented that sulfate reducing bacteria (SRB) are the serious causes of environmental contamination. The anaerobic SRB, leading corrosive microbes, are found in various anoxic environments such as oilfield produced water, freshwater, offshore sediments etc. and acquire their energy by typical enzymatic reduction of sulfate to sulfide. The existence of SRB can create severe environmental and industrial harms, because of sulfide being corrosive, reactive, toxic, and the chief microorganisms in microbiologically induced corrosion.32 On the other hand, these pathogenic bacteria have tremendous abilities in industrial wastewater purification, particularly for removing sulfates and heavy metals from waste effluent.33 Consequently, a biosensing platform with superior sensitivity and selectivity is necessary for precise monitoring of sulfide to regulator microbiological induced corrosion and environmental contamination. Here the biosensing system based on CNTs@CuMn-LDH/GCE is applied for the detection of SRB as sulfide is the characteristic metabolic product of SRB. The centrifugation was done to collect supernatant after 1-4 days of SRB culturing in desulfotomaculum nigrificans culture medium to amass typical metabolic product sulfide in the form of H2S. The concentrations of H2S are detected by amperometric measurements with CNTs@CuMn-LDH/GCE upon the addition of collected supernatant for various days of cultivation. When an aliquot of 100 µL of supernatant is added successively in 15 mL of 0.1 M stirring PBS with subsequent spiking of H2S concentrations, the oxidation current peak goes on increasing correspondingly as shown in Figure 6A-D. It is clear from these figures that as the cultivation time is increased from day 1 to day 4, the concentration of H2S is also enhanced which in turn increases the oxidation current value. Figure 6E expresses the optimal cultivation time span by measuring the peak current responses.



Figure 6. Amperometric responses of CNTs@CuMn-LDH/GCE in the absence (red line) and presence (black line) of SRB supernatant after different time of SRB cultivation such as (A) 1st day, (B) 2nd day, (C) 3rd day and (D) 4th day of incubation, (E) Graph of increasing amperometric current responses with time, (F) Amperometric current records of CNTs@CuMn-LDH/GCE upon the injection of several aliquots of real sample of oilfield produce water, (G) Corresponding linear calibration curve of oilfield produce water sample, (H) Bright field image of A375 cell line, (I) Record of current response on CNTs@CuMn-LDH/GCE in the absence (upper line) and presence (lower line) of A375 cell line upon the addition of 10 μL (40 ng nL−1) of VEGF to each well as stimulator. After first day of SRB cultivation, the minute oxidation current signal to the addition of supernatant might be due to insufficient accumulation of sulfide ions which consequently because of new culture conditions. By continuing the culturing time upto third day, the higher concentration of SRB is resulted that causes enhanced enzymatic process increasing sulfide production which accordingly contributes to higher current responses for spiked volume. The sulfide metabolic rate becomes slower as concentration of microbes reaches its plateau after certain time of cultivation. The maximum population of SRB is obtained at third day of cultivation because the supernatant collected after forth days of culturing shows slightly 14 ACS Paragon Plus Environment

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increased current responses. In addition, the reliability and practical application of CNTs@CuMn-LDH/GCE based biosensing podium have been confirmed by in-situ detection of biogenic H2S efflux in oilfield produced water. The oilfield produced water sample was received from Sinopec Jiangsu Oil Field Industry, Jiangsu and centrifugation was done at 12000 rpm. The amperometric current responses have been presented in Figure 6F upon inoculation of various aliquots of fluid collected from oilfield produced water sample. The obvious current signals for the oxidation of H2S are detected upon injection of each spike of test sample demonstrating the real-time sensing of sulfide for SRB detection. Figure 6G shows the linear relationship between concentrations of oilfield produced water and corresponding oxidation current values. We have calculated the engendered amount of ubiquitous H2S by using regression equation for each 100 µL addition of SRB supernatant and oilfield water produced and also have compared with conventional methylene blue assay (Table S3). The physiological concentrations of H2S hold numerous key properties and function as antioxidants, messenger molecules and apoptosis but its irregular production can be the reason of several chronic attacks. Human melanoma cell line (A375) is cultured and used for real-time monitoring of H2S flux upon the injection of VEGF as stimulator and the corresponding brightfield image of A375 cell line is displayed in Figure 6H. The VEGF is a signaling protein engendered by living cells and possesses noteworthy functions in biological angiogenesis during embryogenesis, growth of skeleton, reproductory processes and it serves as robust stimulating agent to inspire the excretion of cellular H2S efflux.34 Benefitting from tremendous sensitivity conquered, CNTs@CuMn-LDH/GCE is used practically for real-time tracking of H2S released from live cell after being encouraged as depicted in Figure 6I. Upon the addition of 10 μL of VEGF (40 ng nL-1) to every test well of 6-well plate having A375 cells with 80% confluency, substantial oxidation current response of 0.494 μA has been detected. The regression equation has been used to quantify engendered abiotic H2S upon stimulation and measured to be 12.6 µM. While in the absence of cell line, there is no measurable current response, which further authenticates that the excretion of H2S efflux is encouraged by VEGF stimulator. To further ensure the accurateness of CNTs@CuMn-LDH/GCE based biosensor, the result has been compared with conventional methylene blue assay for the detection of H2S concentration which is found to be 13.1 µM. Hence the electrochemical sensing ability of CNTs@CuMn-LDH/GCE based biosensing system possesses almost consistent results obtained by conventional methylene 15 ACS Paragon Plus Environment


blue method in the detection of H2S from A375 cell line. Moreover, CNTs@CuMn-LDH nanohybrids show admirable reproducibility and specificity for the detection of H2S in live cells (Figure S8). Taken together, these results highlight the efficacy of using electrochemical biosensing platform as a viable method for investigating the H2S release.

CONCLUSIONS In conclusion, novel core-shell CNTs@CuMn-LDH hybrid nanomaterial was successfully developed by robust co-precipitation method and used as electrochemical biosensing platform for direct H2S detection. The transitional loading of CuMn-LDH nanosheets on conductive CNTs framework increases the interfacial nanoscale interactions which in turn enhance electron transferring rate and redox process at electrode surface. The enhanced specific surface area enriched with enormous surface active cites, superb electrocatalytic activity of CuMn-LDH, good interfacial collaborations, high charge transport aptitude of CNTs skeleton and unique coreshell morphology synergistically presented excellent electrochemical performance in comparison with other control nanomaterials. The biosensing system consisted of electrode modified with CNTs@CuMn-LDH hybrid architectures demonstrated superior electrocatalytic activity towards H2S oxidation with wide linear range and lowest real detection limit of 0.3 nM (S/N=3). The asconstructed biosensing platform was practically applied in in-situ detection of H2S efflux enzymatically engendered by sulfate reducing bacteria as well as real-time in-vitro tracking of H2S concentrations secreted by human live cells after being motivated upon the inoculation of VEGF stimulator. Therefore, we envision that the structurally integrated CuMn-LDH on the surface of CNTs backbone will offer insight into construction of new type of core-shell hybrid nanoarchitectures for wide spectrum of applications in biosensors, bionanoelectronics and clinical chemistry.

ASSOCIATED CONTENT Supporting information available: Additional details, figures and tables as mentioned in the text. This material is available free of charge via the Internet. Additional experimental details, table, figures showing SEM and TEM images, XRD pattern of control sample and linear relationship of DPV curves 16 ACS Paragon Plus Environment

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CORRESPONDING AUTHORS *E-mail: [email protected] (F. Xiao) *E-mail: [email protected] (H. Liu) NOTES The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported by The National Key Research and Development Program of China (2018YFF0215002), National Natural Science Foundation of China (Project No. U1662114). The Foundation of Hubei Key Laboratory of Material Chemistry and Service Failure (2017), Key Laboratory of Material Chemistry for Energy Conversion and Storage, Ministry of Education (2018). We also acknowledge the support of Analytical and Testing Center of Huazhong University of Science and Technology for XRD, TEM, SEM and XPS (AXIS-ULTRA DLD600W) measurements.

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For TOC only


Hierarchical CNTs@CuMn Layered Double Hydroxide Nanohybrid

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