Gold Nanoparticle

Research Article pubs.acs.org/journal/ascecg

Multifunctional Three-Dimensional Chitosan/Gold Nanoparticle/ Graphene Oxide Architecture for Separation, Label-Free SERS Identification of Pharmaceutical Contaminants, and Effective Killing of Superbugs Stacy Jones, Avijit Pramanik, Rajashekhar Kanchanapally, Bhanu Priya Viraka Nellore, Salma Begum, Carrie Sweet, and Paresh Chandra Ray* Department of Chemistry and Biochemistry, Jackson State University, 1400 J. R. Lynch Street, P.O. Box 17910, Jackson, Mississippi 39217-0510, United States ABSTRACT: Drinking water supplies are now proven to contain pharmaceutical residues, which are becoming a huge global problem. Pharmaceutical residues in water are also responsible for developing drug-resistant superbugs, which has emerged as a significant threat to global health. To tackle the above challenges, the present manuscript reports the development of a chitosan-attached gold nanoparticle conjugated graphene oxide architecture-based multifunctional threedimensional (3D) porous membrane which has the capability for effective separation and label-free surface enhanced Raman spectroscopy (SERS) identification of pharmaceutical contaminants from environmental samples. In the reported design, due to the formation of 3D pores, a multifunctional membrane acted as channels for water passage. On the other hand, due to the presence of several adsorption mechanisms, the hybrid 3D graphene oxide (GO) surface can be used to remove contaminants from water. Due to the presence of a plasmonic nanoparticle-based “hot spot” on the 3D surface, the experimental data presented show that after separation, the 3D SERS substrate has label-free fingerprint identification capability for captured kanamycin antibiotics, the doxorubicin (DOX) chemotherapy drug, and methicillin-resistant Staphylococcus aureus (MRSA) super bugs. The reported data show that due to the presence of antimicrobial nontoxic biopolymer chitosan, the multifunctional 3D architecture can be used for efficient separation, label-free SERS identification, and eradication of MRSA superbugs. A detailed mechanism for label-free identification and killing of super bugs using a 3D membrane have been discussed. KEYWORDS: 3D hybrid graphene oxide, Chitosan based nanoarchitecture, Separation and imaging of pharmaceutical drugs, Capturing and killing of superbugs

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INTRODUCTION According to the World Health Organization (WHO), more than 400 million pounds of antibiotics and other medicines are discarded in the environment each year.1 As per a U.S. Environmental Protection Agency (EPA) study reported in 2013, at least half of all water samples tested by the EPA are positive for 25 drugs.2 Although pharmaceutical pollutants are typically present in the environment at low concentrations, according to the Citizens Campaign for the Environment (CCE), possible health concerns due to the presence of pharmaceutical pollutants are hormone disruption, antibiotic resistance and undesired physiological effects in the society.3 Due to the increase of population every year, pharmaceutical consumption will increase day by day, and as a result, pharmaceutical contaminants, which currently receive minimal consideration by regulators, can be a huge problem for society.3−6 Since the pharmaceutical pollutants are present at a trace level, it is a real challenge to find the amount of a particular pharmaceutical pollutant in the environment.4−11 To tackle the above challenges, the present manuscript reports the © 2017 American Chemical Society

development of a chitosan-attached gold nanoparticle conjugated graphene-oxide architecture-based multifunctional three-dimensional (3D) porous membrane which has the capability for effective separation and label-free surface enhanced Raman (SERS) identification of pharmaceutical contaminants from environmental samples. As reported in Scheme 1, in our design, due to the formation of 3D pores, a multifunctional 3D membrane acted as channels for water passage.12−24 On the other hand, due to the presence of several adsorption mechanisms like the hydrophobic effect, π−π interactions, hydrogen bonds, and electrostatic interactions,25−39 as well as pore-filling, the hybrid 3D GO surface adsorbed pharmaceutical contaminants, which helps to remove contaminants from water. In our design, we have used a plasmonic gold nanoparticle attached 3D membrane for labelfree SERS detection of pharmaceutical contaminants. SERS is a Received: April 30, 2017 Revised: June 14, 2017 Published: June 19, 2017 7175

DOI: 10.1021/acssuschemeng.7b01351 ACS Sustainable Chem. Eng. 2017, 5, 7175−7187

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ACS Sustainable Chemistry & Engineering

Scheme 1. (A) Synthetic Path Used to Develop Chitosan Attached Gold Nanoparticle and (B) Synthetic Path Used to Develop Chitosan Attached Gold Nanoparticle Conjugated Graphene Oxide Architecture Based Multifunctional Three Dimensional (3D) Porous Membrane

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Figure 1. continued

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Figure 1. (A) TEM image showing the morphology of chitosan attached plasmonic gold nanoparticles. (B) SEM image showing the morphology of chitosan attached plasmonic gold nanoparticle conjugated hybrid 3D porous graphene oxide. Inserted high-resolution SEM picture showing the morphology of chitosan attached plasmonic gold nanoparticles inside the 3D membrane. (C) EDX data show the presence of C, O, N, and Au in the developed 3D membrane. (D) IR spectra from chitosan attached plasmonic gold nanoparticle shows the presence of amide-I, amide-II, −C−O−C stretch, −OH stretch, −NH stretch, and −CH stretch IR active vibrational bands. (E) Absorption spectra showing the plasmonic band from chitosan attached gold nanoparticles. The plasmonic band became very broad for the chitosan attached gold nanoparticle conjugated 3D membrane, and it is due to the plasmonic nanoparticle assembly structure in the 3D membrane. (F) SERS spectra from the 3D membrane indicates the presence of D and G bands. (G) N2 adsorption/desorption isotherm for chitosan attached plasmonic gold nanoparticle conjugated hybrid 3D porous graphene oxide indicating the porous structure. Type III isotherms show that chitosan attached plasmonic gold nanoparticle conjugated hybrid 3D porous graphene oxide is composed of macropores. (H) Pore size distributions indicating pore sizes ranging from 400 to 2200 nm and high density at 1300 nm.

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well documented vibrational spectroscopy technique for rapid detection of analytes with chemical specificity intrinsic to vibrational modes.40−57 Due to the presence of a plasmonic nanoparticle based “hot spot” on the 3D multifunctional membrane, after separation, the multifunctional membrane was used for label-free identification of tetracycline antibiotics and doxorubicin chemotherapy drugs. It is now well documented that antibiotic-contaminated water is accelerating the development of drug resistant superbugs, which is a huge challenge for society.1−3,7−11 As per the Center for Global Health, drug-resistant superbugs kill 700 000 people per year, and they are responsible for 10 million deaths per year according to the World Health Organization.7−11 Here, we report the development of a chitosanattached gold nanoparticle conjugated graphene oxide architecture-based multifunctional three-dimensional (3D) porous membrane which has the capability for effective separation, label-free SERS identification, and eradication of a methicillin-resistant Staphylococcus aureus (MRSA) superbug. As shown in Scheme 1, chitosan, an antimicrobial nontoxic biopolymer, is composed of β-(1−4)-linked D-glucosamine and N-acetyl-D-glucosamine.34−39 Due to the cationic nature and strong chelating ability, chitosan is known to exhibit strong antimicrobial activity against various microorganisms.34−39 In our design, due to the presence of antimicrobial nontoxic biopolymer chitosan, the multifunctional 3D architecture was used for efficient separation, label-free SERS identification, and eradication of MRSA superbugs

EXPERIMENTAL SECTION

Chitosan, kanamycin antibiotics, doxorubicin chemotherapy drugs, poly(ethylene glycol) (PEG), KMnO4, graphite, gold chloride, NaBH4, citric acid, nitric acid, and other chmeicals were purchased from Fisher Scientific and Sigma-Aldrich. Multidrug resistant Staphylococcus aureus (MDR-MRSA) superbugs and growth media for growing superbugs were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Development and Characterization of Chitosan Conjugated Plasmonic Gold Nanoparticle. We have developed chitosanattached plasmonic gold nanoparticles using chitosan, acetic acid, and gold chloride, as shown in Scheme 1. For this purpose, 0.05 g of low molecular weight chitosan was dissolved by adding 0.25 mL of glacial acetic acid. Then, we added 5 mL of nanopure water and sonicated vigorously for 30 min to an hour. After that, we removed any undissolved particles by centrifugation. In the next step, we have added 17.25 mL more water and 1000 μL of 10−2 M HAuCl4 solution and started stirring. Then, after some time, the solution turned blue and then immediately to red. Stirring was stopped, and it was left overnight. After that, we used high-resolution JEM-2100F transmission electron microscopy (TEM) and infrared (IR) and absorption spectroscopy to characterize product materials.16,18,21,32,40,41 Figure 1A shows the TEM image for freshly prepared chitosan attached plasmonic gold nanoparticles, which indicates that the particle size is about 25 nm. The infrared (IR) spectra from a freshly prepared chitosan attached plasmonic gold nanoparticle, as reported in Figure 1D, indicates the presence of amide I bands at 1650 cm−1, an amide II band at 1550 cm−1, an OH stretching band coupled with an −NH stretching at 3450 cm−1. We have also observed a C−H stretching band at 2860 cm−1, CH2 bending at 1420 cm−1, and C−O−C stretching bands at 1060 cm−1. The presence of all the above IR bands clearly indicates the presence of chitosan on a gold nanoparticle. The absorption spectra as 7178


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Figure 2. continued

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Figure 2. (A) Absorption spectra from 15 × 10−4 M tetracycline antibiotics in water, before filtering, after filtering using a gold nanoparticle attached 3D GO membrane, and after filtering using a chitosan conjugated gold nanoparticle attached 3D GO membrane. (B) Absorption spectra from 15 × 10−4 M doxorubicin chemotherapy drugs in water, before filtering, after filtering using a gold nanoparticle attached 3D GO membrane, and after filtering using a chitosan conjugated gold nanoparticle attached 3D GO membrane. (C) SERS spectra from tetracycline antibiotics adsorbed on a chitosan conjugated gold nanoparticle attached 3D GO membrane. (D) SERS spectra from doxorubicin chemotherapy drugs adsorbed on a chitosan conjugated gold nanoparticle attached 3D GO membrane. (E) Plot showing tetracycline antibiotics removal efficiency using only 3D GO, GNP attached 3D GO, and a chitosan conjugated gold nanoparticle attached 3D GO membrane. Absorption spectra were used to quantify the removal efficiency. (F) Plot showing doxorubicin chemotherapy drug removal efficiency using only 3D GO, GNP attached 3D GO, and a chitosan conjugated gold nanoparticle attached 3D GO membrane. Absorption spectra were used to quantify the removal efficiency. (G) Plot showing removal efficiency for tetracycline antibiotics and doxorubicin chemotherapy drugs together using a chitosan conjugated gold nanoparticle attached 3D GO membrane, when both drugs (15 × 10−4 M each) were infected into drinking water, Mississippi lake water and Mississippi river water. Absorption spectra were used to quantify the removal efficiency. (H) Concentration dependent SERS spectra from doxorubicin chemotherapy drugs adsorbed on chitosan conjugated gold nanoparticle attached 3D GO membrane. Experimental data showing that 3D substrate based SERS can be used for the detection of DOX even at a concentration of 100 pM. (I) Concentration dependent SERS spectra from tetracycline antibiotics adsorbed on chitosan conjugated gold nanoparticle attached 3D GO membrane. Experimental data showing that 3D substrate based SERS can be used for the detection of tetracycline antibiotics even at a concentration of 100 pM. (J) Plot showing removal efficiency for tetracycline antibiotics and doxorubicin chemotherapy drugs together using a chitosan conjugated gold nanoparticle attached 3D GO membrane, when both drugs (15 × 10−4 M each) were infected into drinking water and different amounts of humic acid. reported in Figure 1E clearly show the presence of a strong plasmon band with λmax at 540 nm. Development and Characterization of Chitosan Conjugated Plasmonic Gold Nanoparticle Attached 3D Hybrid Graphene Oxide Membrane. We have developed chitosan conjugated plasmonic gold nanoparticle attached 3D hybrid graphene oxide

from a chitosan conjugated plasmonic gold nanoparticle and 2D graphene oxide using EDC {1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide} as a cross-linking agent, as shown in Scheme 1A. For this purpose, initially we developed 2D graphene oxide from graphite using a modified Hummers approach, as we have reported before.16,18,21,32,40,41 In the next step, 1 mL of homogeneous 7180


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with λmax ∼365 nm and similarly doxorubicin exhibits well characterized absorption spectra with λmax ∼500 nm, we have used these absorption bands for the measurement of the amount of drugs separated by the 3D membrane. MRSA Superbugs Sample Preparation. We purchased superbug MRSA from the ATCC (ATCC BAA-1707) and then cultured it according to the ATCC protocol, as we have reported before.11,16,18,21,32 Once the bacteria grew to 107 CFU/mL, we diluted using water to vary the concentration of MDR bacteria from 5 to 105 CFU/mL in the infected water solution. Finding the Percentage of Live MRSA Superbugs. We used a colony-countable plate to determine the percentage of live bacteria before and after separation by the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. Experimental details have been reported before. For this purpose, after removal by the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane, we transferred MRSA superbugs to colonycountable plates. Experimental details have been reported before.11,16,18,21,32 We have incubated them for 24 h at 37 °C, and then the colony number was counted with a colony counter (Bantex, Model 920 A).

suspension of GO (4 mg/mL) was mixed with chitosan conjugated plasmonic gold nanoparticles and acid-functionalized poly(ethylene glycol) (PEG), and the mixture was heated at 160 °C for 30 min in an oil bath. After that, we used EDC as a cross-linking agent to develop the 3D porous architecture by using the coupling chemistry between the −CO2H group of 2D graphene oxide and the −NH2 group from a chitosan conjugated plasmonic gold nanoparticle. In the presence of SH-PEG, due to the strong binding of the Au−S bond, PEG binds with the gold nanoparticle and allows the −NH2 group from chitosan to bind with 2D graphene oxide, as shown in Scheme 1. Next, the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide was spin-casted to develop 3D pores with a size of 5 × 5 cm2. Next, we used a Hitachi 5500 ultra-high-resolution scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDX) analysis, Raman and absorption spectroscopy, and Brunauer− Emmett−Teller (BET) nitrogen adsorption analysis to chracterize the 3D membrane. Experimental details have been reported before.16,18,21,32,40,41 Figure 1B shows the SEM characterization of the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane, which indicates that our membrane is an interconnected 3D network with a pore size of 2−3 μm. Inserted highresolution SEM characterization shows that the chitosan conjugated plasmonic gold nanoparticles are in an assembly structure in the 3D membrane. Figure 1C shows the EDX mapping data which indicate the presence of Au, N, C, and O in the 3D SERS substrate. This proves that the chitosan conjugated plasmonic gold nanoparticles are attached on the 3D membrane. Absorption spectra reported in Figure 1E show a very broad plasmon band for chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane, which is mainly due to the assembly structure formation by chitosan conjugated plasmonic gold nanoparticle in the 3D membrane. Raman spectra from the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane, as reported in Figure 1F, indicate a strong D band at ∼1347 cm−1 and a G-band at ∼1626 cm−1.20−32 The observed stronger D band in comparison to the G band clearly indicates that the surface modification extent for graphene oxide is very high in our 3D membrane. 3D Membrane Characterization. We have performed a Brunauer−Emmett−Teller (BET) experiment to find the specific surface area, pore volume, and pore density for our developed chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. As reported in Figure 1G, our reported experimental N2 adsorption/desorption data show type III isotherms, which indicate that chitosan attached plasmonic gold nanoparticle conjugated hybrid 3D porous graphene oxide is composed of macropores. BET analysis indicates that the specific surface area for the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane is 780 m2 g−1, with a pore volume of 0.730 cm3 g−1. As shown in Figure 1H, the average pore diameter was around 1300 nm. We also measured the water flux of our chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. For this purpose, we collected the permeated water through the membrane using an electronic balance. From the experimental data, we calculated the water flux of our chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane as 232.8 L m−2 h−1 bar−1. SERS Measurement. For the Raman experiments, we used fiber optics probe based SERS using a continuous wavelength DPSS laser with 2 mW of power at 785 nm of light as the excitation light source. Experimental details of the SERS measurement have been reported before.40,41,43,46,50,51 For SERS data collection from chitosan conjugated plasmonic gold nanoparticle attached 3D hybrid graphene oxide substrate, a miniaturized QE65000 spectrometer from Ocean optics has been used. Finding the Percentage of Tetracycline Antibiotics and Doxorubicin Chemotherapy Drugs Separated by 3D Membrane. To measure the amount of tetracycline antibiotics and doxorubicin chemotherapy separated by the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane, we used absorption spectra, as reported in Figure 2A and B. Since tetracycline antibiotics exhibit well characterized absorption spectra

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RESULTS AND DISCUSSION To understand whether the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane can be used for the separation and identification of tetracycline antibiotics and doxorubicin chemotherapy drugs, we performed filtration of a 100 mL water sample containing 15 × 10−4 M of tetracycline antibiotics and doxorubicin chemotherapy drugs separately. We used absorption spectra for finding the removal amount for antibiotics and doxorubicin chemotherapy drugs after filtration using a 3D membrane as reported in Figure 2A. As shown in Figure 2A,B and E,F, our data clearly show that 92% doxorubicin chemotherapy drugs and 88% tetracycline antibiotics have been captured by the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. This high removal efficiency for tetracycline antibiotics and doxorubicin chemotherapy drugs using a chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane can be due to several possible mechanisms, and these are (1) adsorption via π−π interaction between graphene oxide and tetracycline antibiotics/doxorubicin chemotherapy drugs, (2) adsorption via hydrophobic interaction between graphene oxide and tetracycline antibiotics/doxorubicin chemotherapy drugs, (3) adsorption via strong electrostatic interaction between chitosan and tetracycline antibiotics/ doxorubicin chemotherapy drugs, (4) adsorption via Hbonding between chitosan and tetracycline antibiotics/doxorubicin chemotherapy drugs, and (5) removal via pore-filling mechanism due to the porous structure of the 3D membrane. To understand how the presence of chitosan conjugation helps to remove the drugs, we have also performed the same experiment with only 3D GO and GNP attached 3D GO without chitosan. As reported in Figure 2E,F, the removal efficiency increased more than 25% due to the presence of chitosan, which clearly indicates that strong electrostatic interaction between chitosan and drugs helps to remove drugs abruptly. Next, to understand whether the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane can be used for label-free identification of pharmaceutical drugs via surface enhanced Raman spectroscopy (SERS), we performed SERS measurement on a 3D membrane 7181


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Figure 3. (A) SEM image showing MRSA has been captured by the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. (B,C) Colonies of bacteria showing the amount of live MRSA in water (B) before filtration using the chitosan conjugated plasmonic gold 7182


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ACS Sustainable Chemistry & Engineering Figure 3. continued

nanoparticle attached 3D graphene oxide membrane and (C) after filtration using the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. (D) SERS spectra from MRSA adsorbed on the chitosan conjugated gold nanoparticle attached 3D GO membrane. (E) Plot showing MRSA removal efficiency using only 3D GO, GNP attached 3D GO, and chitosan conjugated gold nanoparticle attached 3D GO membrane. A reverse transcription polymerase chain reaction (RTPCR) was used to quantify the removal efficiency. (F) Concentration dependent SERS spectra from MRSA adsorbed on a chitosan conjugated gold nanoparticle attached 3D GO membrane. Experimental data shows that 3D substrate based SERS can be used for the detection of MRSA even at a concentration of 5 CFU/mL. (G) Plot showing MRSA removal efficiency using a chitosan conjugated gold nanoparticle attached 3D GO membrane in the presence of albumin serum protein and MRSA culture media. A reverse transcription polymerase chain reaction (RTPCR) was used to quantify the removal efficiency.

after filteration. To eliminate the D and G Raman band contribution in Raman spectra from pharmaceutical drugs on a 3D substrate, we have subtracted the Raman spectra of drugs adsorbed on the 3D membrane from the spectrum for only the 3D membrane. As a result, all the observed Raman bands as shown in Figure 2C and D could be assigned to vibration bands for pharmaceutical drugs.53−57 As reported in Figure 2C, in the case of tetracycline, we observed an amide-I band and CO stretching vibration band at 1645 and 1595 cm−1, respectively. Similarly, we observed C−C and C−N stretching vibrational bands at 1230 and 1360 cm−1. We also observed SERS bands due to cyclohexene ring vibration and breathing. Similarly, as shown in Figure 2D, the Raman spectrum from DOX exhibits amide-I, −CO stretch, CO deformation modes at 1641, 1575, and 440 cm−1. Similarly, we have observed a ring stretch, a −CN stretch, and secondary alcohol Raman bands as shown in Figure 2D. By comparison of Figure 2C and D, we found that −CO in-plane deformation and secondary alcohol Raman bands are unique for DOX, which we have not observed for tetracycline antibiotics. Similarly, Raman bands due to an aromatic ring are unique for tetracycline, which we have not observed for DOX. All the above results indicate that chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane-based SERS can be used for label-free fingerprint sensing of DOX and tetracycline. As shown in Figure 2H, the concentration dependent SERS data from DOX adsorbed on the chitosan conjugated gold nanoparticle attached 3D GO membrane clearly indicate that 3D substrate based SERS can be used for the detection of DOX even at a concentration of 100 pM. Similarly, Figure 2I shows that 3D substrate based SERS can be used for the detection of tetracycline antibiotics even at a concentration of 100 pM. Next, to find out whether our chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane can be used for the separation of drugs from the environmental sample, we used Mississippi Lake water and Mississippi River water that was infected with 15 × 10−4 M of tetracycline antibiotics and doxorubicin chemotherapy drugs simultaneously. After that, we performed filtration of a 100 mL spiked Mississippi River water sample using our chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. As reported in Figure 2G, the removal efficiency for drugs from an environmental sample using our developed chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane is about the same as what we have observed in the case of drinking water. The above data indicate that the drug removal efficiency remains mostly unaltered with a change of slight pH and the presence of salt and other metal ions. Next, to find out whether our developed chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane can be used for the separation of drugs in the

presence of humic acid, we used drinking water infected with 15 × 10−4 M of tetracycline antibiotics and doxorubicin chemotherapy drugs as well as different amounts of humic acid simultaneously. As reported in Figure 2J, the removal efficiency for drugs remains about the same in the presence of different amounts of humic acid. Next, to understand whether our developed chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane can be used for the separation, identification, and killing of MRSA superbugs, we added 1.6 × 105 colonyforming units (CFU)/mL of MRSA in drinking water. In the next step, we performed gentle shaking of the mixture of drinking water and MRSA for 100 min, and then we used the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane for filtering water to separate MRSA superbugs. After removal of superbugs from infected water using a chitosan conjugated membrane, the removal efficiency was determined using a reverse transcription polymerase chain reaction (RT-PCR) technique. We have also verified the result using a colony plating technique on LB agar, as shown in Figure 3. RT-PCR and colony counting data as reported in Figure 3B, C, and E indicate that about 100% of superbugs can be removed using a chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. The high efficiency of superbug removal using a chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane is due to the fact that the interaction between the positively charged chitosan and the negatively charged microbial cell wall leads to wrapping of superbugs on the surface of chitosan.34−39 Similarly, we and others have shown that graphene oxide also interacts with bacteria via a mechanical trapping mecahanism.16,20−32 As shown in Figure 3A, our reported data clearly show that MRSA has been trapped in 3D pores, when MRSA infected water was filtered using a chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. We believe that both a wrapping and trapping mechanism are involved for the removal of 100% of the superbugs from water. To demonstrate that chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane-based SERS can be used for label-free identification of trapped MRSA superbugs, we performed SERS measurement on the 3D membrane after being filtered. To eliminate the D and G Raman bands’ contribution in Raman spectra from MRSA superbugs on the 3D substrate, we subtracted the Raman spectra of MRSA superbugs trapped by the 3D membrane from the spectrum for only the 3D membrane. As a result, all the observed Raman bands as shown in Figure 3D could be assigned to vibration bands for MRSA superbugs. As reported in Figure 3D, experimental data show that MRSA exhibits SERS bands for amide-I, −CH2 deformation, lipid C−C deformation, and the C−N aromatic for protein and fatty acids.40,41,52−54 To 7183


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ACS Sustainable Chemistry & Engineering understand how the presence of chitosan conjugation helps to remove the MRSA superbugs, we also performed the same experiment with only 3D GO and GNP attached 3D GO without chitosan. As reported in Figure 3E, the removal efficiency increased around 50% due to the presence of chitosan, which clearly indicates that strong electrostatic interaction between the positively charged chitosan and the negatively charged microbial cell wall helps to remove MRSA abruptly. As shown in Figure 3F, the concentration dependent SERS data from MRSA adsorbed on the chitosan conjugated gold nanoparticle attached 3D GO membrane clearly indicates that 3D substrate based SERS can be used for the detection of MRSA even at a concentration of 5 CFU/mL. Next, to understand whether a chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane can be used for the separation of MRSA in the presence of biomedical related substances, we performed an MRSA removal experiment in the presence of 500 mg of albumin serum protien. As reported in Figure 3G, the removal efficiency for MRSA is about the same as what we have observed in the absence of albumin serum protein. The removal efficiency decreases only 2−3% in the presence of 500 mg of albumin serum protein. Similarly, as reported in Figure 3G, the removal efficiency for MRSA is decreased only ∼3% in the presence of MRSA culture media. For this experiment, we have used a 5:1 concentration of water and culture media. Since MRSA is a superbug, disinfection of MRSA after separation is necessary to avoid spreading it to others. To find out whether the captured superbug by the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane is dead or not, initially we washed the 3D membrane surface thoroughly with water several times. In the next step, we used a colony plating technique that determines the amount of live MRSA. As reported in Figure 4C, around 100% of superbugs were killed when they have been filtered using chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. To understand better, we also performed the same experiment using a 3D graphene oxide based membrane and a gold nanoparticle attached chitosan based 3D membrane. As reported in Figure 4A, B, and E, only 10% of superbugs were killed when we used a 3D grpahene oxide based membrane. On the other hand, around 75% of superbugs were killed when we used a gold nanoparticle attached chitosan based 3D membrane. We also measured killing efficiency using only chitosan, and we observed around 74% killing efficiency for MRSA. All of the above results clearly show that chitosan presence is necessary to kill MRSA superbugs. It is now well documented that chitosan exhibits strong antimicrobial properties.34−39 As shown in Figure 4D, due to the porous structure, MRSA was trapped in a porous chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. Under this condition, there will be a strong interaction between the positively charged chitosan and the negatively charged cell wall via coordinating lipopolysaccharide.34−39 The above strong interaction will help for the leakage of intracellular constituents. During this process, superbug cell lysis happens due to the disruption of the permeability barrier of the outer membrane.34−39 Now in the case of only the graphene oxide 3D membrane, we observed 10% superbug killing, and it is due to the fact that during trapping, graphene oxide can induce perturbation to some extent on the superbugs’ cellular membrane.16,20−32 So in this case, a superbug gets killed due to the membrane damage via

Figure 4. (A−C) Colonies of bacteria showing the amount of live MRSA on the membrane, (A) after filtration using only a graphene oxide based 3D membrane, (B) after filtration using chitosan conjugated plasmonic gold nanoparticle based 3D membrane and (C) after filtration using the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane. (D) SEM image showing the MRSA are captured by the 3D membrane. (E) Plot showing % of live MRSA when superbug infected water was filtered using only 3D GO, GNP attached chitosan based 3D membrane, chitosan attached 3D GO membrane, and chitosan conjugated gold nanoparticle attached 3D GO membrane. The colony plating technique was used to quantify the amount of live superbugs. We performed this experiment five times, and the average has been reported.

mechanical wrapping. Since in the case of the chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane both electrostatic and perturbation via mechanical wrapping occur simultaneously, we have observed 100% killing efficiency. Similarly, as shown in Figure 4E, 99% killing efficiency was observed for the chitosan conjugated 3D GO membrane without gold nanoparticles, where both killing mechanisms, electrostatic interaction via chitosan and perturbation via mechanical wrapping by GO, work simultaneously. 7184



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AUTHOR INFORMATION

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Research Article

REFERENCES

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In conclusion, here we have reported the development of a chitosan attached gold nanoparticle conjugated graphene oxide architecture based multifunctional three-dimensional (3D) porous membrane with a specific surface area of 780 m2 g−1, a pore volume of 0.730 cm3 g−1, and an average pore diameter of 1300 nm, which can be used for effective separation and label-free SERS identification of pharmaceutical contaminants from environmental samples. We have shown that due to the presence of several adsorption mechanisms like a strong electrostatic interaction, π−π interaction, hydrophobic interaction, H-bonding, and pore filling, our chitosan conjugated plasmonic gold nanoparticle attached 3D graphene oxide membrane is capable of capturing and separating 92% of the doxorubicin chemotherapy drugs and 88% of the tetracycline antibiotics from an environmental sample. We have demonstrated that the 3D membrane not only captured drugs from the environmental sample but it also can be used for label-free detection of pharmaceutical drugs using a Raman fingerprint. Our reported data also show that the same chitosan attached gold nanoparticle conjugated graphene oxide architecture based multifunctional 3D porous membrane can be used for effective separation, label-free SERS identification, and eradication of the MRSA superbug. Due to the presence of a strong electrostatic interaction between the positively charged chitosan and the negatively charged microbial cell wall, as well as the interaction of graphene oxide with bacteria via a mechanical trapping mecahanism, the 3D membrane can be used for 100% separation of superbugs from the water sample. Reported experimental results show that due to the presence of plasmonic nanoparticles, the 3D membrane can be used for label-free identification of captured superbugs. Due to the strong electrostatic interaction via coordinating lipopolysaccharide, chitosan has the capability to cause the leakage in the intracellular constituents and graphene oxide has the capability to cause membrane damage via mechanical wrapping; our 3D membrane not only has the capability to capture superbugs but it also can kill 100% of superbugs after capturing. Since chitosan can be made by treating the chitin shells of shrimp and GO can be prepared from naturally present crystalline allotrope of carbon, one can easily develop a large scale chitosan attached graphene oxide architecture based 3D porous membrane using a low-cost synthesis method. We believe that after proper large scale and low cost design, our multifunctional 3D porous membrane has enormous potential for the separation and identification of drugs and superbugs from an environmental sample, as well as for killing superbugs.

Corresponding Author

*Fax: +16019793674. E-mail: [email protected]. ORCID

Paresh Chandra Ray: 0000-0001-5398-9930 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Ray thanks NSF-PREM grant # DMR-1205194, NSF CREST grant # 1547754, and NSF RISE grant # 1547836 for their generous funding. 7185


Research Article

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