Development of a Sensitive Diagnostic Device Based on Zeolitic

Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/ascecg

Development of a Sensitive Diagnostic Device Based on Zeolitic Imidazolate Frameworks‑8 Using Ferrocene−Graphene Oxide as Electroactive Indicator for Pseudomonas aeruginosa Detection Saeed Shahrokhian*,†,‡ and Saba Ranjbar† †

Department of Chemistry and ‡Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Azadi Avenue, Tehran 11155-9516, Iran

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S Supporting Information *

ABSTRACT: Since Gram-negative bacteria have a predominant role in nosocomial infections, there are high demands to develop a fast and sensitive method for diagnosis of bacteria in clinical samples. To address this challenge, we designed a novel electrochemical biosensor based on aptamers immobilized in engineered zeolitic imidazolate Framework-8 (ZIFs-8) via EDC-NHS chemistry. Cyclic voltammetry and electrochemical impedance spectroscopy techniques were conducted to monitor the electrochemical characterization. With respect to unique π−π interactions between aptamer and graphene oxide (GO), the differential pulse voltammetry technique was applied with ferrocene-graphene oxide (Fc-GO) as an electroactive indicator for the detection of Pseudomonas aeruginosa (P. aeruginosa). In the presence of P. aeruginosa, the configuration of the aptamer showed a change and Fc-GO was released from the electrode surface. On the basis of the signal-off strategy, the proposed biosensor exhibits a wide linear dynamic range (from 1.2 × 101 to 1.2 × 107 CFU mL−1) with a low detection limit. The results reveal that the fabricated aptasensor has high potential applicability in the field of monitoring disease therapy and controlling the safety of the clinical sites. KEYWORDS: Zeolitic imidazolate framework-8, Graphene oxide, Electrochemical biosensor, Pathogen diagnosis, Pseudomonas aeruginosa, Differential pulse voltammetry

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based on affinity reagents such as antibodies,9 aptamers,10 antibacterial peptides,11 and bacteriophages.12 By considering a high binding affinity with long-term stability and cost favorability, biosensors based on aptamers have received the most attention in recent decades.13,14 Electrochemical-based aptasensor holds great promise for real-time monitoring of pathogens by low-cost assay, convenient miniaturization, and ease of signal translation.15,16 The main challenge to improve the electrochemical biosensing technology is designing a suitable modified platform to effectively immobilize biorecognition elements.17,18 Metal−organic frameworks (MOFs), a class of ultraporous hybrid materials formed by connecting organic linkers with inorganic nodes, have been widely interesting in various fields, particularly in sensing due to their high surface area with excellent porosity, designable pore size, and high thermal and chemical stability.19 Zeolitic imidazolate frameworks (ZIFs) consist of transition-metal cations and imidazole-based ligands as a subclass of MOFs, rapidly expanding due to better stability in aqueous solutions with the environmentally friendly synthesis in room temperature.20,21 Moreover, porous ZIF

INTRODUCTION Hospital-acquired infections (HAIs) have been listed among serious hospital difficulties that influence the quality of health care. In particular, for patients admitted into the intensive care unit (ICU), accident, burn, and surgery unit, this risk is in a high due to their underlying disease and impaired immunity.1 A report published in 20052 stated that HAIs are the sixth leading cause of death in the United States that imposed 5−10 billion dollars in cost annually. Aerobic Gram-negative bacteria such as Pseudomonas aeruginosa (P. aeruginosa) have a main role in HAIs including pneumonia and the urinary tract.3 To reduce the infections and compensate for the complications, numerous antibacterial agents are prescribed against P. aeruginosa.4 However, dramatic increases in antibiotic resistance led to developing reliable and fast methods for diagnosing the pathogenic bacteria in hospital and clinic sites.5 Various gold standard methods have been applied for the detection of pathogenic bactera during these years. These protocols, e.g., culture colony, biochemical tests, immunoassay, and polymerase chain reaction (PCR), show high selectivity and accuracy6,7 but unfortunately suffer from labor-intensive, time-consuming, and several tedious purification and enrichment steps.8 Recent efforts have been focused to extend an effective method to diagnosis of bacteria whole cells with high sensitivity, great selectivity, fast response time, and cost-effective technology © XXXX American Chemical Society

Received: March 6, 2019 Revised: June 17, 2019 Published: July 7, 2019 A

DOI: 10.1021/acssuschemeng.9b01314 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

The lyophilized powders of Pseudomonas aeruginosa and Escherichia coli O157:H7 aptamers with amine modification were purchased from Bioneer Corp. (South Korea) with BIO-RP purification and the following sequence according to previous literature obtained by the Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX) process.29,30 P. aeruginosa Aptamer Sequences. 5′-NH2 CCC CCG TTG CTT TCG CTT TTC CTT TCG CTT TTG TTC GTT TCG TCC CTG CTT CCT TTC TTG-3′ Escherichia coli O157:H7 Aptamer Sequences. 5′-NH2 CCG GAC GCT TAT GCC TTG CCA TCT ACA GAG CAG GTG TGA CGG-3′ Sterile distilled water (SD water) was used to prepare ssDNA aptamers stock solution (267.6 pmol μL−1) and 10 times diluted stock solution for fabrication of aptasensor. Methods. The voltammetric experiments were conducted using a Metrohm potentiostat/galvanostat analyzer model 797VA. Electrochemical impedance spectroscopy (EIS) measurements were carried out by using an Autolab PGSTAT 204 (controlled by Nova 2.2 software), which performed with a frequency between 5 kHz and 100 mHz and a 5 mV rms sinusoidal modulation under the open-circuit potential (OCP) condition in the presence of a redox probe (0.1 M KCl containing 5 mM Fe(CN)63−/4−). During the electrochemical measurements, a cell containing three electrodes including a glassy carbon working electrode (d = 2.0 mm, Azar Electrode Co.), an Ag/ AgCl (saturated KCl) reference electrode, and a Pt wire auxiliary electrode was used. In cyclic voltammetry (CV) the scan rate was adjusted 100 mV s−1, and in differential pulse voltammetry (DPV) the modulation amplitude was 50 mV and the step potential was 6 mV. Cleaning the electrode surface and preparing the modifier suspensions were done with an ultrasonic bath (KODO model JAC 1002, Korea). A PerkinElmer spectrophotometer (Lambda 25) was applied for concentration adjustment of cultured bacteria. The field emission scanning electron microscopy technique (FESEM, MIRA3 TESCAN) with an energy-dispersive X-ray spectroscopy (EDS) detector was used to characterize the surface morphology and elemental composition of the prepared materials. Transmission electron microscopy (TEM) images were obtained with PHILIPS CM200 to show the hollow structure of HZIFs-8. Nitrogen adsorption/desorption measurements were done at 77 K at using a micrometrics model ASAP2010 sorptometer. Prior to N2 adsorption, the samples were evacuated at 373 K under vacuum condition. Specific surface areas were determined by multipoint Brunauer− Emmett−Teller (BET) theory from nitrogen adsorption data. The thermal stability of the synthesized ZIF crystals was checked by a TGA (Mettler Toledo 851) analyzer from room temperature to 873 K at a heating rate of 10 K/min under N2 atmosphere. An X-ray diffractometer (GBC MMA) was used to investigate the crystal phases of the prepared materials in the 2θ range from 5° to 90° using Cu Kα irradiation. A PerkinElmer FT-IR spectrophotometer (spectrum 100) was applied to evaluate the chemical structure of the synthesized materials with prepared KBr pellets. Dynamic light scattering (DLS) analysis was conducted with a Zetasizer Nano ZS from Malvern Instrument equipped with a He−Ne laser (λ = 632.8 nm) in the backscattering detection mode at ambient temperature to estimate the size distribution of ZIFs-8 and HZIFs-8 samples. P. aeruginosa Cultivation and Plate Counting. The as-used bacteria strains including P. aeruginosa, E. coli O157:H7, Staphylococcus aureus, Salmonella typhimurium, and Shigella f lexneri were grown by overnight shaking at 37 °C in Luria−Bertani medium (LB). The bacteria concentration was adjusted in an optical density of 0.67 at 600 nm (OD600) and examined by McFarland Turbidity Standard.31 In the following, the bacteria cells were washed with sterile PBS (0.1 M, pH = 7.4) three times by centrifuge at 6000 rpm. Finally, the desired dilution series were resuspended in fresh PBS and can be kept at 4 °C for 1 week before use. Each experiment was done under aseptic conditions to make sterile media. The exact number of colony-forming unit per milliliter (CFU mL−1) of the cultured (24 h, 37 °C) P. aeruginosa colonies were counted in the LB agar plate as a standard method.

structures, especially hollow porous structures, can provide a large surface area with most abundant active sites, low density, a lower distance for mass transfer, and favorable open structure. However, unfortunately, the synthesis of nanomaterials with controllable hollow structure and morphology is still a challenge. Chemically the in situ method by using (poly)phenolic acids as an etching agent is one way to prepare ZIFs with high porosity and hollow structure that also provided surface functionalization. Interaction of phenolic acid derivatives with the ZIF leads to changing the surface of ZIFs from a hydrophobic to a hydrophilic nature by releasing the proton that is attached to the ZIF structure and creating mesoporous holes inside it. The attached phenolic acids prevent the further etching and destroying of crystals; therefore, a hollow porous structure ZIFs (HZIFs) is obtained with abundant active sites containing carboxylic acid groups to immobilize larger amounts of targets.22 Among the different electrochemical methods,23−25 differential pulse voltammetry (DPV) employing electroactive indicators provided a simple and effective strategy for monitoring clinical and biological targets. The high surface area with π conjugation ability makes 2D graphene and its oxides (GO) more attractive to prepare a suitable platform for immobilizing organic and inorganic molecules, which promote the development of graphene-based electroactive indicators.26 GO also has a strong potential for the adsorption of aptamers with effective π−π interactions to use as an indicator in aptasensor fabrication.27 Because of the stable and reversible electrochemical response, ferrocene (Fc), a sandwich compound in which the Fe atom is accommodated between two cyclopentadienyls, has been used as an appropriate redox molecule to functionalized graphene oxide for preparing a stable electroactive indicator in the biosensing strategy.28 In the present work, the HZIFs via synergistic etching and surface functionalization were synthesized by using phenolic acid at room temperature, which exhibits well-defined hollow structures and abundant carboxylic acid groups in the shell to better immobilize −NH2 aptamer with EDC-NHS chemistry. Differential pulse voltammetry (DPV) with ferrocene-graphene oxide (Fc-GO) as a redox indicator was used to translate the interaction between aptamer and whole cell bacteria. In the presence of P. aeruginosa, the interaction of Fc-GO with the aptamer was inhibited by binding aptamer to specific epitopes of bacteria and forming a unique 3D structure containing a bacteria−aptamer complex. Under the optimized conditions, the designed biosensor based on a signal off strategy was applied as an executable device for the detection of P. aeruginosa in human urine samples.

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EXPERIMENTAL SECTION

Materials. Zinc nitrate hexahydrate [Zn(NO3)2·6H2O], 2methylimidazole (C4H6N2), tannic acid (C76H52O46), copper acetate monohydrate [Cu(CH3COO)2·H2O], glucose (C6H12O6·H2O), sodium hydroxide (NaOH), nitric acid (HNO3), ferrocene [Fe(C5H5)2], N-hydroxysuccinimide (NHS), N-(3-(dimethylamino)propyl)-N-ethylcarbodimidehydrochloride (EDC), potassium ferrocyanide, potassium ferricyanide, ethanol, methanol, acetone, potassium chloride, and ceftazidime (CFZ) were purchased from Merck and Sigma-Aldrich companies in analytical grade, which were used without further purification. Graphene oxide (GO) was ordered from graphene supermarket (Graphene Laboratories, Inc. USA). Deionized water (DI, 18.2 MΩ) was used in the whole experiments. All bacterial strains were kindly provided by the Microbiology Department of the Pasteur Institute of Iran. B


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ACS Sustainable Chemistry & Engineering Synthesis of HZIFs-8 as an Electrode Modifier. ZIF-8 nanoparticles with a polyhedron structure were synthesized in an aqueous system according to Pan et al.32 In brief, 0.239 g of Zn(NO3)2·6H2O and 5.68 g of 2-methylimidazole (2-MIm) were separately dissolved in two beakers containing 2 and 20 mL of DI water. Then the 2-MIm solution was added to the zinc solution under constant stirring for 5 min to form a milky-colored suspension. The resulting suspension was kept for 6 h at room temperature without disruption. Finally, the white product was collected with centrifuging, washed with DI water, and dried at 60 °C overnight. In-situ chemical etching with tannic acid (TA, as an etching agent) was done to synthesize the HZIFs-8.22 In this procedure, Zn−N coordination bonds of ZIFs were broken by protons (H+) released from TA and new N−H bonds and coordinated Zn−TA complex were formed. For this purpose, 5 mg of the synthesized ZIF-8 nanoparticles was mixed with 5 mL of TA solutions (5 g L−1) and aged for 30 min. After that the light yellow HZIF-8 nanoparticles with a polyhedron structure containing abundant carboxylate groups were collected by centrifugation, washed with methanol/DI water, and dried at 60 °C. Synthesis of Fc-GO as an Electroactive Indicator. First, the ethanolic solution of ferrocene (Fc, 50 mg in 50 mL) was added to GO suspension (50 mg dispersed in 50 mL of DI water) for 1 h under sonication, and the obtained suspension was shaken slightly for 24 h. Next, the nanocomposite of Fc-GO was acquired by centrifuging the final suspension at 6000 rpm for 5 min. The Fc-GO suspension, as an electroactive indicator for translating the formation of aptamer− bacteria complex, was prepared in ethanol solution (2 mg/mL). Synthesis of Cu2O Particles as an Antibacterial Agent. According to the previous literature,33 first 3 g of Cu(CH3COO)2· H2O was dissolved in 20 mL of DI water under constant stirring at 70 °C for 2 min. Then a dark precipitate appeared by adding 10 mL of sodium hydroxide solution (9 M) under stirring for 5 min after addition of 0.6 g of D-glucose powder to the prepared precipitation followed with a constant stirring speed at 70 °C for 60 min; a red solid product was collected by centrifugation, washed with DI water/ ethanol (equal ratio), and dried in an oven at 60 °C. Biosensor Fabrication. Before the modification procedure, the surface of the GCE was pretreated with sandpaper followed by a 0.1 μm alumina slurry, sonicated in ethanol/DI water, and washed with DI water for 1 min. Afterward, 4 μL of the HZIFs-8 suspension was drop cast on the GCE followed by drying in 50 °C. Then 20 μL of EDC-NHS (40 mM:50 mM) was added on the surface of HZIFs-8/ GCE for 2 h to activate the carboxylic groups of the HZIFs-8. Following, 15 μL of the amino-functionalized P. aeruginosa aptamer (26.7 pmol μL−1) was immobilized at the EDC-NHS/HZIFs-8/GCE at 4 °C overnight (Scheme. 1A). Owing to the low density and high porous structure of HZIFs-8, a large accessible surface area with lots of free carboxylic acid groups was provided to immobilize aminemodified aptamers. Finally, in order to remove unbounded aptamers, the fabricated aptasensor was rinsed thoroughly with DI water several times and cycled between 0.0 and 1.0 V (vs Ag/AgCl, with scan rate 100 mV s−1) in PBS five times to acquire a stable background current. HEX.8.0.0 software was performed to predict the binding behavior of aptamer and P. aeruginosa. The aptamer acts as a guest for the specific epitope of P. aeruginosa to form aptamer−bacteria complex via van der Waals forces, hydrogen bonding, and electrostatic interactions. The results of these interactions are depicted in Figure S1. Also, the secondary structure of the aptamer was imaged with mfold (Figure S2, Supporting Information). P. aeruginosa Detection. P. aeruginosa detection was done based on a signal-off strategy by the DPV technique in the potential range from −0.3 to 0.6 V (vs Ag/AgCl) by employing Fc-GO as a redox indicator (Scheme 1B). In order to tag Fc-GO on the electrode, the as-prepared Apt/HZIFs-8/GCE was immersed in Fc-GO suspension (2 mg/mL) for 60 min. The DPV signal was recorded in PBS (0.1 M, pH = 7.4) to confirm the successful attachment of Fc-GO. π−π interactions between the aptamer and graphene are responsible for the attachment of Fc-GO to the electrode, which further creates an electrochemical signal to translate the interaction of the whole cell− aptamer. The nonspecific adsorbed Fc-GO on the electrode was

Scheme 1. Schematic Illustration of (A) Aptasensor Fabrication and (B) Pseudomonas aeruginosa Signal-off Detection

released from the aptasensor by immersing it in pure PBS for 20 min. In the following, the as-prepared aptasensor was incubated in P. aeruginosa (with various concentrations) in the optimized conditions. Due to a change in the conformation of aptamer-tagged Fc-GO in the presence of P. aeruginosa, Fc-GO was removed from the surface of the electrode which is accompanied by a change in the effective electron transfer to the electrode (Figure S3). The analytical signal and LOD were calculated using the following equations

ΔIp = Ip,Fc‐GO(Aptamer_PBS) − Ip,Fc‐GO(Aptamer_P . aeruginosa) (1) LOD = 3S b/m

(2)

where Sb is the blank standard deviation, m is the slope of the calibration curve, and ΔIp is determined based on Fc-GO peak current changes before and after the presence of P. aeruginosa. Four human urine samples were collected from healthy volunteers (25−35 years old, men and women) and spiked with different concentrations of P. aeruginosa without any pretreatment to check the performance of the proposed method. The designed aptasensor was applied to analyze the urine samples using the electrochemical assay mentioned above, and the control experiment was done to remove the unwanted effects by incubation of aptasensor in urine samples without P. aeruginosa as a blank.

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RESULTS AND DISCUSSION Physicochemical Characterization of the Synthesized Modifier. The surface morphology of ZIFs-8 and HZIFs-8 was investigated by the FE-SEM technique. According to Figure 1A, the ZIF-8 particles show nanocrystal structures with a hexagonal facet and an average particle size of approximately 250 nm (approved by DLS analysis, Figure S4A). Chemically in situ etching of ZIFs-8 in tannic acid was controlled with FESEM images and DLS analysis and confirmed that the obtained particles have similar shape and size to the initial ZIF-8 crystals (Figures 1B and S4B). Hollow porosity of HZIFs-8 was followed with the TEM image (Figure 1C) by observing low contrast inside the particles. Furthermore, the presence of −COOH groups at the surface of ZIFs-8 after the etching process was confirmed with increasing the amount of oxygen in C


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Figure 1. FE-SEM images of (A) ZIFs-8 and (B) HZIFs-8. (C) TEM images of HZIFs-8. EDS analysis of (D) ZIFs-8 and (E) HZIFs-8.

Figure 2. Results of (A) zeta-potential changes, (B) FTIR spectra, (C) XRD pattern, (D) N2 sorption curves, and (E) TGA analysis obtained for (a) ZIFs-8 and (b) HZIFs-8.

N−H bond. For HZIFs-8, the same pattern was maintained and new bands at 887, 1584, and 1697 cm−1 observed, revealing the presence of the out-of-plane bending vibrations of the isolated hydrogen in the benzene ring, OH bending vibration, and CO stretching vibration of the carbonyl group at the surface of HZIFs-8 (Figure 2B). XRD patterns (Figure 2C) of initial and hollow porous ZIFs8 to confirm the crystal planes were studied that well-defined and almost similar pattern shows the same crystal structure for both. However, observing the low intensity of HZIFs-8 XRD peaks suggested that the etched sample has a lower crystallinity due to breaking Zn−N bonds from ZIFs in the presence of TA.22 The surface area and pore-size distribution of ZIF-8 and

the EDS pattern of HZIFs-8 compared to ZIFs-8 (Figure 1D and 1E). In agreement with this result, Figure 2A shows a considerable decrease in the zeta-potential value for HZIFs-8 compared to ZIFs, which means the surfaces of ZIFs were properly covered with TA, which in turn provides carboxylic functional groups on the HZIFs-8. In this context, FT-IR spectra also demonstrated the successful synthesis of HZIFs-8 from ZIFs-8 nanoparticles. The FT-IR spectrum recorded for ZIFs-8 clearly indicated the characteristic peaks at 900−1300, 1308, 1427, and 1573 cm−1, and a broad peak at 3200−3500 cm−1 corresponded to in-plane bending of the ring, entire ring stretching, CO stretching vibration, and stretching of the D


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Figure 3. (A) Cyclic voltammograms (scan rate, 100 mVs−1) and (B) Nyquist plots of 0.1 M KCl containing 5 mM Fe(CN)63−/4− in each step of aptasensor fabrication for 1.2 × 107 CFU mL−1 P. aeruginosa detection.

Figure 4. (A) Cyclic voltammograms (scan rate, 100 mVs−1) and (B) Nyquist plots of 0.1 M KCl containing 5 mM Fe(CN)63−/4− recorded for (a) Apt/HZIFs-8/GCE, (b) Fc-GO/Apt/HZIFs-8/GCE, and (c) Fc-GO/Apt/HZIFs-8/GCE incubated in 1.2 × 105 CFU mL−1 P. aeruginosa.

weight loss steps in the temperature range from 200 to 340 °C (10%) and 340 to 380 °C (8%) are results of evaporation of moisture and embedded solvent and also decomposition of the tannic acid as a shell around ZIFs. Electrochemical Characterization of Designed Aptasensor. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were recorded in the presence of a redox probe (0.1 M KCl containing 5 mM Fe(CN)63−/4−) to characterize aptasensor fabrication (Figure 3). As can be seen, a remarkable decrease in the redox peak currents together with an increase in the charge transfer resistance (Rct) is recorded for GCE after modification with ZIFs-8 due to the ZIF nanocrystals low conductivity. These trends were maintained with the etching of ZIFS-8 in TA and synthesis of HZIFs-8. The observed behavior can be related to the presence of a lot of carboxylic groups in the HZIFs-8 structure that repelled the negatively charged probe from the surface of the electrode. Next, through activation of carboxylic acid groups at the surface of HZIFs-8 with EDC-NHS as a crosslinking agent, due to reducing the negative charge of the surface, the peak currents are increased and consequently Rct decreased. Coupling of amine-modified aptamer with HZIFs-8 via EDC-NHS and the effect of cross-linking agent are illustrated in detail in Scheme S1 and Figure S6. Next, by immobilization of the aptamer at the surface of EDC-NHS/

HZIF-8 crystals were examined by N2 adsorption/desorption isotherms (Figure 2D). Observing type-I behavior for pure ZIFs-8 indicates the microporous crystal structure; however, the type-IV isotherms with a hysteresis loop in the range of P/ P0 = 0.4−0.9 obtained for etched ZIFs-8 in TA confirmed the presence of mesoporosity in the HZIF-8 crystals. Furthermore, high adsorption capacities at high relative pressure (P/P0 > 0.9) demonstrated the coexistence of micro- and mesopores in hollow porous ZIFs. The etched ZIFs-8 shows a Brunauer− Emmett−Teller (BET) surface area of 1078.2 m2 g−1, which is smaller than that of the initial ZIFs-8 (1206.8 m2 g−1), which is the result of partial amorphization of ZIFs-8 in the etching process. The pore size distributions calculated by the BJH method are presented in Figure S5. The pore sizes were narrowly distributed between 1 and 2 nm, indicating that ZIF8 nanoparticles had a highly uniform pore structure. However, the pore size distributions widened, and larger pore diameters distinctly increased after etching. The thermal gravimetric analysis (TGA) data recorded for controlling thermal stability (Figure 2E) exhibit a weight-loss step (18%) before 180 °C for ZIFs-8 corresponding to water or other guest molecules removal and a long plateau in the temperature range 200−600 °C that indicated the high thermal stability of ZIF-8 crystals. The gradual weight loss is observed at two steps for hollow porous ZIFs-8. The first and second E


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Figure 5. Results of (A) FE-SEM, (B) elemental mapping, (C) EDS, and (D) FT-IR analysis obtained for Fc-GO as an electrochemical indicator.

HZIFs-8/GCE, the redox peak currents are decreased and Rct increased related to the presence of phosphate groups in the aptamer backbone that repelled the negatively charged probe from the electrode surface. Finally, incubation of the aptasensor in 1.2 × 107 CFU mL−1 of P. aeruginosa suspension leads to decreasing the peak currents and increasing the Rct that confirmed formation of aptamer−P. aeruginosa complex, creating the steric hindrance for diffusion of the redox probe to the electrode surface. Characterization of Fc-GO as an Electroactive Indicator. In the proposed strategy ferrocene (Fc) acted as an electroactive indicator and graphene oxide was used as a carrier for Fc, which also has the potential to interact with the aptamer via π−π stacking. The electron transfer kinetics of FcGO-modified aptasensor was evaluated by CV and EIS methods. Owing to the high electrical conductivity of FcGO, by immersing the aptasensor in Fc-GO suspension and in the presence of the redox probe (0.1 M KCl containing 5 mM Fe(CN)63−/4−), the charge transfer resistance (Rct) is decreased and consequently the peak current increased. Afterward, incubation of Fc-GO/Apt/HZIFs-8/GCE in 1.2 × 105 CFU mL−1 of P. aeruginosa led to the formation of aptamer−P. aeruginosa complex and removal of the Fc-GO from the electrode surface, which caused an increase in the Rct and decrease in the peak current for the redox probe (Figure 4). The crucial role of GO in aptasensor response was checked by incubation of the aptasensor in Fc suspension. The results declared that without using GO stable and effective interaction between the aptamer and Fc was not established, and therefore, a very weak DPV signal has resulted for the aptasensor in Fc suspension (Figure S7). The physicochemical characterization of Fc-GO was evaluated in detail by using FE-SEM, elemental mapping,

EDS, and FT-IR analysis. The FE-SEM image (Figure 5A) shows that the ferrocene chemically modified GO nanosheets are well exfoliated. The elemental mapping image of individual elements was used to confirm the decoration of GO with Fc by showing the distribution of iron atoms on GO nanosheets (Figure 5B). Moreover, the presence of Fe, C, and O was confirmed in the Fc-GO backbone with EDS analysis (Figure 5C). Also, the FT-IR characteristic peaks corresponding to the Ring-Titt of torsional vibration of Fc, the asymmetric stretching vibrational of x-CON (x = C or Fc), the CO stretching of −CON, and the stretching of O−H appeared at 481, 1003, 1653, and 3379 cm−1, respectively (Figure 5D). These observations confirmed the successful synthesis of FcGO using a chemical approach.34 Also, the stability of Fc-GO as an electroactive indicator was checked by recording CVs of the aptasensor incubated in FcGO for 50 cycles in PBS. The CV curves of Fc-GO display a pair of redox peaks corresponding to the Fc/Fc+ redox couple at +0.2 V, which indicates a strong interaction between Fc-GO and the aptamer at the surface of the electrode (Figure S8). Optimization of the Experimental Parameters. By considering the predominant role of modifier amount in the aptamer immobilization for P. aeruginosa incubation, different volumes of HZIFs-8 suspension (1−5 μL) were cast on the GC electrode. The results (Figure S9) showed that increasing the volume of HZIFs-8 suspension to 4 μL leads to preparing a stable film with a high amount of carboxylic acid groups to immobilize −NH2-functionalized aptamers. Further increase in the volume of modifier suspension has no remarkable improvement in the DPV response of Fc-GO. The effect of indicator incubation time on the aptasensor efficiency is shown in Figure S10. The results revealed that by increasing the Fc-GO incubation time to 60 min, the DPV signal of the aptasensor increased and after this time reached a F


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Figure 6. (A) DPV responses of aptasensor in the presence of Fc-GO for different concentrations (2.1 × 107−2.1 × 101 CFU mL−1) of P. aeruginosa. (B) Corresponding linear calibration curve of ΔIp versus logarithm of P. aeruginosa concentration.

Figure 7. (A) DPV signal of Fc-GO, (B) cyclic voltammograms, and (C) Nyquist plots in the presence of 0.1 M KCl containing 5 mM Fe(CN)63−/4− for (a) aptasensor, aptasensor incubated in (b) ceftazidime-treated P. aeruginosa (1.2 × 105 CFU mL−1), (c) Cu2O nanoparticlestreated P. aeruginosa (1.2 × 105 CFU mL−1), and (d) 1.2 × 105 CFU mL−1 live P. aeruginosa cells.

plateau. This observation is the result of gradual π−π interaction between the aptamer and GO in ∼60 min. Therefore, a time of 60 min for indicator incubation was chosen for further experiments. The decrease in the DPV response of Fc-GO tagged aptamer in the presence of P. aeruginosa was controlled as a sign of forming aptamer−P. aeruginosa complex. To optimize the time needs for binding aptamer with P. aeruginosa; the prepared aptasensor was incubated in 1.2 × 105 CFU mL−1 bacteria suspension at a different time. According to the results in Figure S11, a significant decrease was observed in DPV signal until 20 min as a result of releasing the Fc-GO from the electrode surface. After that the DPV signal of Fc-GO is almost constant; therefore, 20 min was set as an optimum value. Analytical Characterization and Performance of Aptasensor. The response sensitivity of the aptasensor was tasted by exposing in Fc-GO, PBS, and serial dilutions of P. aeruginosa (from 1.2 × 109 to 1.2 × 10 °CFU mL−1) followed by monitoring the DPV signal of Fc-GO in the absence and presence of P. aeruginosa. As shown in Figure 6, capturing P. aeruginosa by the aptasensor caused the release of Fc-GO from

the electrode; therefore, the DPV signal of Fc-GO decreased by increasing the concentration of P. aeruginosa. A good linear relationship was obtained between ΔIp Fc‑GO and the logarithm of P. aeruginosa concentration in the range from 1.2 × 101 to 1.2 × 107 CFU mL−1. Moreover, a LOQ of 12 CFU mL−1 and LOD of 1 CFU mL−1 was determined for the fabricated aptasensor (each experiment was three times repeated, and LOD was calculated by 3Sb/m, where Sb is the blank standard deviation and m is the slope of the calibration curve). Repeatability of the aptasensor response was determined by 5 times repetitive detection of PBS (as a blank) and 1.2 × 103 and 1.2 × 106 CFU mL−1 P. aeruginosa using the same electrode by recording the Fc-GO peak current. The relative standard deviations (RSD) of 5.27%, 2.85%, and 3.0% for the mentioned solutions confirmed good precision of fabricated aptasensor in the analysis of bacteria samples. The reproducibility was also investigated by checking the DPV responses of Fc-GO for five independent aptasensor in PBS and 1.2 × 103 and 1.2 × 106 CFU mL−1 of P. aeruginosa, and RSD values were calculated as 6.9%, 4.9%, and 7.8%, respectively. G


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Table 1. Comparison of the Analytical Performance between Presented Biosensors for P. aeruginosa Detectiona proposed sensor AuNPs/Ppy-COOH/GSPE glove coated with SPE carboxyl graphene/GCE CCLP-Au NPs/GCE ANDPA/Glu-AgNPs Bt-PEG thiol/PEG thiol on the Au nanotriangles AuNPs SPIO HZIFs-8/GCE

LOD

biorecognition layer

detection strategy

LDR

pyoverdine marker DPV SWV ECL LSV optical LSPR

1−100 μm 1−100 μm 5−50 μm 1.4 × 102−1.4 × 106 CFU mL−1 3.1 × 101−7.3 × 107 CFU mL−1 0.1−8 CFU mL−1 101−103 CFU mL−1

0.33 μm 0.33 μm 0.33 μm 56 CFU mL−1 900 CFU mL−1 1.5 CFU mL−1 1 CFU mL−1

ref

bacteriophage antibody antibody aptamer

37 38 39 40 13

colorimetric/SERS MRSw DPV

102−106 CFU mL−1 102−106 CFU mL−1 1.2 × 101−1.2 × 107 CFU mL−1

50/20 CFU mL−1 50 CFU mL−1 1 CFU mL−1

aptamer aptamer aptamer

41 42 this work

36

a

LDR: Linear dynamic range. LOD: Limit of detection. AuNPs: Gold nanoparticles. Ppy-COOH: Carboxylic polypyrrole. G-SPE: Graphene-based printed electrode screen. SPE: Screen-printed electrode. SWV: Square-wave voltammetry. GCE: Glassy carbon electrode. ECL: Electrochemiluminescence. CCLP: Calcium cross-linked pectin. LSV: Linear sweep voltammetry. ANDPA: (R)-4-(anthracen-9-yl) −6- (naphthalen-1-yl)1,6- dihydropyrimidine-2-amine. Glu-AgNPs: Glucose-stabilized silver nanoparticles. SERS: Surface-enhanced Raman spectroscopy. SPIO: superparamagnetic iron oxide. MRSw: magnetic relaxation switch. Bt-PEG: Biotinylated polyethylene glycol. LSPR: Localized surface plasmon resonance. HZIFs-8: Hollow porous zeolitic imidazolate Framework-8. DPV: Differential pulse voltammetry.

compared to the live one with the same concentration. Furthermore, CV (Figure 7B) and EIS (Figure 7C) confirmed that changes in the redox peak currents and charge transfer resistance of the aptasensor in the presence of the redox probe (0.1 M KCl containing 5 mM Fe(CN)63−/4−) for dead cells are considerably smaller than the live one. It means the binding behavior of aptamer with live and dead P. aeruginosa is not the same because of the difference in the membrane integrity of live and dead cells. Exposing P. aeruginosa cells in the antibacterial bacterial agents lead to deforming and denaturation of protein and lipopolysaccharide layers on the cell membrane, which restrict the affinity of aptamer to the dead bacteria cells. Nonspecific binding of aptamer with denatured cells and also some remaining viable bacteria in the suspensions are two reasons for weak responses which were recorded for antibacterial-treated P. aeruginosa.35 The synthesized copper oxide particles were characterized by FE-SEM, EDS, FT-IR, and XRD analysis. The Cu2O particles with decahedron morphology were obtained (Figure S15 A) and confirmed by elemental composition, crystal phase, and chemical structure data (Figures S15B−D). In recent years, impressive efforts have been done toward extending effective and reliable biosensors for detection of P. aeruginosa in real samples. Table 1 reports some of these results to compare the analytical performance of proposed methods. According to the table, various detection methods and biorecognition elements were employed against P. aeruginosa. Cernat et al.36 and Ciui et al.37 introduced two different electrochemical biosensors for detection of pyoverdine as a certain P. aeruginosa marker that show good linear dynamic range with low LOD. However, by considering the short time and direct analysis without needing enrichment and purification, detection of the whole cell is most considerable for bacteria compared to the specific markers. P. aeruginosa whole cell detection is performed by using various molecular recognition elements, namely, bacteriophage,38 antibody,39,40 and aptamer.13,41,42 Among the different specific elements, aptamers are more interesting due to their high stability, low cost, ease of synthesis, and low dependence on pH and temperature that encourage us to use them for the fabrication of biosensor. Our proposed aptasensor has a potential to detect P. aeruginosa in a wide linear dynamic range to 7 orders of

The ability of the aptasensor to discriminate between target and nontarget bacteria was also studied by incubation in P. aeruginosa, E. coli O157:H7, S. aureus, S. typhimurium, and S. f lexneri (with the same concentration of 107 CFU mL−1). The results confirmed that the aptasensor prefers an acceptable selectivity toward P. aeruginosa in comparison to the nontarget bacteria (Figure S12). Two different experiments were performed as the control for evaluating the role of aptamer and affinity of aptasensor to P. aeruginosa.by recording the DPV signal of Fc-GO. In the first case, the modified electrode based on HZIFs-8/GCE without aptamer immobilization was incubated in Fc-GO suspension. Due to the nonspecific and weak adsorption of graphene oxide at the surface of the electrode, the initial signal of Fc-GO is much less than that when aptamer is present in the sensor components. In the following, easily removing the redox indicator from the surface of the electrode observed by incubation of sensor in PBS or P. aeruginosa confirmed the role of aptamer in this sensing strategy. In the second experiment, the HZIFs-8/GCE was modified with E. coli O157:H7-specific aptamer and applied for detection of P. aeruginosa. The obtained results revealed that variation in the DPV signal of Fc-GO is limited compared to the aptasensor fabricated with specific P. aeruginosa aptamer (Figure S13). Regeneration of the aptasensor was controlled by dissociating aptamer from P. aeruginosa in 2 M NaCl for 1 h. Results of regeneration for 3 cycles for the detection of 1.2 × 105 CFU mL−1 P. aeruginosa indicated a good performance for the aptasensor in several times of application (Figure S14). Longterm stability of the biosensor is also one of the main factors in practical applications, which were investigated for the proposed aptasensor; 93.4% of the initial signal was preserved after 2 weeks of holding in the refrigerator at 4 °C. The proposed aptasensor was tested with live and dead P. aeruginosa cells to demonstrate its ability of differentiating between them. P. aeruginosa cells were exposed to antibacterial agents including ceftazidime (antibiotic drug) and copper oxide particles, and DPV, CV, and EIS signals were recorded in the presence of live and antibacterial-treated P. aeruginosa. In Figure 7A, the DPV signals of Fc-GO indicator show smaller changes for the aptasensor incubated in 1.2 × 105 CFU mL−1 of dead P. aeruginosa (treated with both antibacterial agents) H


Research Article

ACS Sustainable Chemistry & Engineering magnitude, similar to the results obtained by Krithiga et al.,39 which used antibody to modify calcium cross-linked pectin/ gold nanoparticles for biosensor fabrication. However, in the case of LOD, the performance of our sensor is better and comparable with the sensor introduced by Sundaram et al.40 and Hu et al.13 Colony plate counting was controlled as a reference method to validate the aptasensor response. A 10−7 time diluted sample of P. aeruginosa was coated on the LB agar plate and incubated at 37 °C for 20 h. Then the cultured colony was counted, and the mean of three times replication was calculated as 139 CFU mL−1. In this investigation, a relative error of 13.67% was obtained for the response of the fabricated electrode compared to the reference method. The feasibility and reliability of the designed aptasensor for the detection of P. aeruginosa in real samples were evaluated by a recovery experiment. Under the optimized conditions, the aptasensor was used to analyze healthy human urine samples (volunteer men and women, 25−35 years old), spiked with different concentrations of P. aeruginosa. The samples were analyzed, and the recovery results are given in Table S1. Obtaining good recoveries (in the range of 81.25−118.33%) confirmed the applicability of the fabricated aptasensor in clinical analysis.

ORCID

Saeed Shahrokhian: 0000-0003-3138-6578 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the support of this work by the Research Council and the Center of Excellent for Nanostructures of Sharif University of Technology, Tehran, Iran. They are also immensely grateful to Ms. Yeganeh Mirzaei for her contribution proofreading the English grammar of the manuscript.

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CONCLUSIONS Herein, we designed an electrochemical device for detection of whole cell bacteria, P. aeruginosa as a model, based on the aptamer immobilization on the surface of engineered ZIFs-8 via carboimide cross-linking. Diagnosis of bacteria was monitored by the DPV signal of Fc-GO as an electroactive indicator. Fc-GO can interact with aptamer and can be adsorbed on the surface of aptasensor via π−π stacking and be removed from the electrode surface in the presence of P. aeruginosa. The proposed whole-cell sensing platform was capable to detect P. aeruginosa in a wide linear dynamic range (from 1.2 × 101 to 1.2 × 107 CFU mL−1) and LOQ 12 CFU mL−1 with good sensitivity, selectivity, repeatability, and stability in the response. Finally, the fabricated aptasensor showed good potential for direct diagnosis of P. aeruginosa in human urine samples.

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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/acssuschemeng.9b01314. Schematic of interaction between aptamer and HZIFs with EDC-NHS chemistry; data of HEX 8.0.0 and mfold software; detection strategy of fabricated aptasensor; DLS analysis and pore size distribution of as-synthesized materials; influence of EDC-NHS in the response of aptasensor; influence of graphene oxide in the electroactive indicator; stability of Fc-GO; optimization parameters; selectivity; affinity and regeneration of proposed aptasensor; surface characterization of Cu2O particles; table of recovery test for real sample (PDF)

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REFERENCES

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Development of a Sensitive Diagnostic Device Based on Zeolitic

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