- Email: [email protected]
Monolayer graphene chemiresistive biosensor for rapid bacteria detection in a microchannel
Journal Pre-proof
Monolayer graphene chemiresistive biosensor for rapid bacteria detection in a microchannel Weiqi Zhao , Yunyi Xing , Yang Lin , Yuan Gao , Mengren Wu , Jie Xu PII: DOI: Reference:
S2666-0539(20)30001-1 https://doi.org/10.1016/j.snr.2020.100004 SNR 100004
To appear in:
Sensors and Actuators Reports
Received date: Revised date: Accepted date:
21 December 2019 4 February 2020 12 February 2020
Please cite this article as: Weiqi Zhao , Yunyi Xing , Yang Lin , Yuan Gao , Mengren Wu , Jie Xu , Monolayer graphene chemiresistive biosensor for rapid bacteria detection in a microchannel, Sensors and Actuators Reports (2020), doi: https://doi.org/10.1016/j.snr.2020.100004
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Highlights
Microfluidic chemiresistive biosensors are fabricated using monolayer graphene, gold nanoparticles, streptavidin-antibody system, and PDMS.
The biosensor captures E. coli bacteria and performs sensing via gate-free resistance response.
The developed biosensor is proved to be simple, rapid and sensitive for bacteria detection.
1
Monolayer graphene chemiresistive biosensor for rapid bacteria detection in a microchannel
Weiqi Zhao, Yunyi Xing, Yang Lin, Yuan Gao, Mengren Wu and Jie Xu* Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA E-mail: [email protected]
Graphical abstract
2
Abstract Bacteria, such as Escherichia coli (E. coli), can cause food poisoning and serious diseases. In fact, E. coli has emerged multiple times as a severe public health concern in recent years. Therefore, it is important to detect bacteria like E. coli in simple, rapid and sensitive approaches. In this study, a gate-free chemiresistive biosensor based on monolayer graphene (MG) is proposed to detect E. coli, thanks to the high sensitivity stemming from the anti-E. coli antibody-coated graphene. The immobilization of the antibodies is performed via streptavidin and biotin conjugation on the graphene surface. Compared to conventional bacterial biosensors, the proposed chemiresistive biosensor captures the E. coli bacteria on the surface of the sensor and performs the detection through electric readouts, other than optical signals. That is the resistance measured in the biosensor increases along with the increase of concentration of the bacteria. To further extend the capabilities of this biosensor and provide a controllable test flow, a microchannel is integrated on the graphene surface. The results show that the developed chemiresistive biosensor is able to detect E. coli in trace concentration down to 12 cfu ml-1, indicating an excellent sensing performance. The proposed monolayer graphene based chemiresistive biosensor clearly manifest its advantages of low-cost, ease of fabrication, portability and good sensitivity, as well as the potential for rapid in-situ bacterial detection. Keywords – Bacteria, Escherichia coli (E. coli), Monolayer graphene (MG), Chemiresistive Biosensor, Concentration, Microchannel
3
Introduction Bacteria accompany our lives, but sometimes they will cause problems in our health. For example, Escherichia coli (E. coli), which usually colonizes human intestines right after birth [1], inevitably affects human beings throughout their entire life spans. The discovery of E. coli can be dated back to 1885 by Theodor Escherich, and it was originally considered as a healthy mutualism bacterium helping human beings with synthesis of vitamin K2 [2]. In 1996, scientists found that E. coli can also cause food poisoning in human beings, as evidenced by a worldwide outbreak of food poisoning occurred in Scotland [3]. In addition, E. coli infections can cause diarrhea and lead to life-threatening conditions, such as kidney failure, high blood pressure, chronic kidney disease, and neurologic problems. To minimize the impact of E. coli on human society, efficient diagnosis of the bacteria is of utmost importance. At present, a variety of methods have been developed to detect and determine the concentration of E. coli. For instance, one of the most straightforward methods is through cell culture, in which E. coli is cultured on a petri dish for 24 h at 37 °C in an incubator. By means of counting the number of E. coli colonies, the concentration of E. coli can be determined [4]. Nonetheless, this method usually requires long preparation time. Therefore it has been gradually substituted by other approaches. For example, nucleic acid-based polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) technology could give rise to a much more accurate result in a shorter time [5-7]. Nevertheless, they are not satisfactory for rapid detection as they are costly, time consuming and require immobile laboratory settings. Recently, various electrochemical based biosensors for the detection of E. coli pathogen has been developed [8]. For example, anodic particle coulometry technique has been combined with cyclic voltammetry (CV), and the concentration of the E. coli decreases as the absorption spectra mean decreases [9]. Moreover, nanomaterials such as gold nanorods and gold nanoparticles (AuNPs) have also been incorporated into sensing assays to further enhance the specificity and sensitivity of detection [10]. For example, Bhalla and coworkers have shown that the gold nanoparticles can increase the sensitivity for biosensor significantly. Specifically, the gold nanoparticles helped the detection of cardiac troponin I (cTnI) at a concentration as low as 0.2 ng mL−1, which is much better than ELISA tests of 4.3 ng mL−1 [11]. Depending on the function of the gold nanoparticles, it can be linked to aptamer for capturing target bacteria. In order to find the adaptor on the E. coli membrane for aptamer, the amplified single-strand DNA need to be translated based on the DNA array of E. coli, in which the concentration of E. coli can be obtained [12]. However, the DNA screening of aptamer is relatively difficult, and the specificity provided by aptamers is often limited. In this study a chemiresistor biosensor based on monolayer graphene is developed with electrical readouts for E. coli detection. Instead of using aptamers, we choose to use antibody-streptavidin system together with AuNPs for high specificity and high sensitivity bacteria recognition. Streptavidin has been proved to be effective when it is linked to gold nanoparticles for biosensing [13]. The sensing element in our system is monolayer graphene (MG). Graphene is widely used in biosensors because of its special electrical properties [14]. Graphene can transfer 15000 cm2 Vs-1 electrons at room temperature while the resistance is only around 10-6 ohm.cm. Moreover, multiple studies of 4
monolayer-graphene have confirmed their enormous stiffness and ultrathin thickness [15-17]. Thereby monolayer graphene with large contact area is naturally suitable for providing abundant binding sites for sensing purpose. Graphene’s electric properties, such as conductivity, is very sensitive to its environment, not to mention chemical bindings on its surface. Hence, we immobilize integrated AuNPs and streptavidin-antibody system on the graphene surface for E. coli capturing and monitor the electrical signals generated. Gold nanoparticles were spread over monolayer graphene to produce conductivity, meanwhile, the electrode was produced from gold nanoparticle which can increase the sensitivity of biosensors [18]. As observed in the experiments, when the system binds with E. coli, the resistance of the monolayer graphene is increased. Therefore, the bio-signals of E. coli binding are converted into electrical signals, and the concentration of E. coli is then verified. It is worth mentioning that both graphene and gold also have a special function in antibacterial, which makes the device safer to operate for pathogen-related applications [19]. With the development of microfluidic technology, different types of lab-on-a-chip devices with in-channel chemical sensors have been developed. For instance, the microfluidic system based on single-walled carbon nanotubes [20] and based on enzyme assays [21] have been integrated for detecting glycerol concentration. In addition, a microchannel system also can be used in the glucose detection [22]. Research activities and advances in lab-on-a-chip devices have revolutionized the field of biosensing research, since sensing in a microfluidic environment often benefits from stable and precise sample control, parallel and multiplexed assay capabilities, and scaling-up potential. By nature, electrochemical sensors are easily and effectively to integrate with microfluidic platforms because of their simple structure. In this paper, the developed biosensor is integrated into a lab-on-a-chip device for the detection of different concentration of E. coli solution.
1. Material and methods 1.1. Materials Monolayer graphene coated on a square substrate of Si/SiO₂ was purchased from Graphenea, Inc. Gold/palladium nano-particle target was purchased from Ted Pella, Inc. Phosphate buffered saline (PBS, 0.1 mol l-1, pH 7.4) with 0.1% Tween 20 was purchased from Sigma Aldrich Inc. Streptavidin was purchased from AnaSpec, Inc. E. coli K12 ER2925 were purchased from New England Biolab, Inc. Biotinylated anti-E. coli (from rabbit) antibody was purchased from abcam, Inc. Goat anti-rabbit IgG (Heavy & Light Chain) antibody (Atto 488) was purchased from Antibody-Online, Inc. The tryptic soy broth and tryptic soy agar for medium were purchased from Thermo Fisher Scientific, Inc. 1.2. Device fabrication The overall detection system is illustrated in Fig. 1(a). Monolayer-graphene substrate with a size of 1 cm by 1 cm was first scrutinized to ensure a good quality. Afterwards, two electrodes were coated on the surface of graphene substrate with gold/palladium alloy target (99.99% Au:Pd, 60:40 ratio) supported by a low pressure argon gas chamber (0.05 mbar) using a Au/Pd Polaron 5
sputter coater E5100 series II. As shown in Fig. 1(b), a predesigned mask was fixed on the upper surface of the substrate, after which the sputter coater was used to deposit two 15 nm thick Au/Pd continuous films as electrodes (for 10 minutes). After stripping the mask, the sputtering coater was used for a second time, but the second time coating was on the full surface of the graphene substrate and was only for 10 seconds. This way, the center of graphene substrate had been coated with nanoparticle stratum granulosum as Au particle linker. After the preparation of the electrodes, two PDMS layers were added onto the substrate to create a microchannel. The first layer was the microchannel layer of PDMS which was used to cover the top of graphene piece. The width, length, and the depth of the microchannel are 800μm, 8mm and 1mm, respectively. The dimensions are chosen because they can be made conveniently using low-cost technology. Specifically, the PDMS layers were created via standard soft lithography [23, 24]. Specifically, the mold of the microchannel was fabricated on a polymethyl methacrylate (PMMA) plate by a micro-milling machine (CNC Mini-Mill/3 PRO). The PDMS pre-mixture was then mixed, degassed, and poured onto the mold, and kept in an oven for 8 min at 75℃. While the PDMS was still a little bit sticky, it was adhered onto the graphene, and punched the injection and extraction holes on the two sides of the channel. After that, the graphene was placed with the PDMS layer down on the plate. Then the second layer PDMS liquid was poured onto the substrate and sealed the graphene substrate into the PDMS. The whole device was placed in the oven for 20 mins at 75 ℃. The two grooves were cut on the two sides of the electrodes, which were used to gain electric contact on the electrodes for electric measurements (Fig. 2). Finally, the microchannel was rinsed with deionized water (DI water), in order to remove impurities in the microchannel. Afterwards, the microchannel was injected and incubated overnight with 15µl streptavidin (1mg ml-1) at 4 ℃. After rinsing with buffer (PBS containing 0.005% Tween 20) and DI water thoroughly, the graphene was treated by 15 μl of 0.5 mg ml-1 biotinylated anti-E. coli antibodies for 2 hours, followed by rinsing with DI water. After the fabrication of devices, they can be used immediately for the detection of bacteria. In our experiment, a series of E. coli K12 solutions with different concentrations were injected into the microchannel for 5 mins consecutively for E. coli detection and measurements. If the devices are not to be used immediately, it is recommended to seal an antibody stabilizer PBS buffer in the microchannels for preserving the proteins and antibodies for a relatively long time, and the devices should be kept in a refrigerator (2-8 ℃). 1.3. Bacteria preparation The original E. coli K12 was cultured in the tryptic soy broth solution and incubated in the incubator for 16 hours at 37 ℃ with 246 RPM stir speed. The bacterial population were maximized, and then the initial concentrations of bacteria samples were obtained by serial dilution. The tryptic soy agar was used in a petri dish to culture E. coli, in order to verify the initial count of the E. coli, using the so-called plate count method.
6
Figure 1. (a) The structure of the PDMS microchannel. The PDMS microchannel was fixed on the monolayer-graphene with electrodes. Under the PDMS microchannel is the structure of the biosensor in the center of the microchannel. (b) The sputter coater was used to coat two sides of electrodes. The mask was covered on the upper surface of the graphene. 1.4.Electrical measurement Electrical measurements were performed on monolayer-graphene biosensor using a micropositioner with a Keithley 4200 source meter system at room temperature. The sensing signal of the device was recorded by LabView software monitoring the change between two electrodes in the current (I) for a given source voltage (U) from 0 V to 2 V when the device was exposed to different concentrations of E. coli solution. The scanning electron microscopy (SEM) images were obtained using a JSM 6320F SEM. Raman spectra was obtained on a micro-Raman spectrometer excited by 532 nm laser excitation. Fig. 2 shows the actual graphene biosensor 7
integrated in a PDMS microchannel. Two grooves on the two sides of the electrodes were used to get access to the electrodes.
Figure 2. A photo of the proposed microfluidic chemiresistive biosensor.
2. Results and discussion 2.1. Characterization of monolayer-graphene The SEM image of the MG is depicted in Fig. 3(a), which shows an area in between the two electrodes of the sensor. As observed, the graphene film is continuous, uniform, and dominantly single-layered structure across the entire surface. Raman spectra measurements were performed to further confirm the presence of the high-quality MG layer. As shown in Fig. 3(b), the MG displays at two prominent peaks at 1580 cm-1 and 2676 cm-1, corresponding to the well-documented G and 2D, respectively [25, 26]. The fact that the 2D peak is greater than the G peak indicates the presence of a defect-free MG layer on the chemiresistor sensor, which is also in accordance with the evidence from the SEM image (Fig. 3(a)).
(a)
(b)
Figure 3. (a) A small area of the MG-based chemiresistive sensor in between the Au/Pd electrodes was characterized by SEM; (b) Raman spectra of the MG chemiresistive sensor. 8
2.2. Characterization of linker The linkers were applied on the graphene substrate to capture the E. coli, which then causes a change in resistance in MG for detection. The primary linkers are Au nanoparticle, Streptavidin and antibody on the graphene. Streptavidin is the most widely used analogue of avidin [27]. The avidin-biotin affinity interaction was approximately 103 to 106 times higher than an antibody-antigen interaction [28]. Therefore, application of streptavidin in this biosensor enables very stable linking (compared to antibody-antigen). Because streptavidin can protect the biotinyl esters from hydrolysis, but avidin can augment this hydrolysis [29]. Au nanoparticles and streptavidin are biologically specific binding. These functional groups of water-soluble nanoparticles are usually carboxylic acids, which stabilize nanoparticles by electrostatic repulsion and can be used to conjugate other molecules to particles [30]. In order to verify these linkers (Au nanoparticle, streptavidin and anti-E. coli antibody) can be linked together very well, we used labelled secondary antibody to verify that the anti-E. coli had fixed on the surface. The Au nanoparticles were coated on a glass slide. Meanwhile a circle was drawn on the glass slide, and the streptavidin and anti-E. coli solution were dropped on this area respectively. After that, PBS solution was applied to rinse the slide surface three times. The secondary antibody with fluorescence under the laser microscope was observed in the circled area only. Therefore, it was confirmed that these linkers had been jointed together and the other linkers which were not linked had been rinsed out. 2.3. Results and discussion In the bacteria-antibody binding system, the amount of the bacteria-antibodies bond being produced during the sensing process directly affects the resistance of the MG. It is reported that the membrane of nature biological cells shows a resistance of 102-105 Ω cm-1 [18, 31]. When the bacteria cells attach to the surface of graphene film, the formation of bacteria-antibody conjugation could produce kinetics barrier for the electron transfer process, which may induce more distinct changes in the carrier hole density in graphene film through polarization of cell-surface charges, intracellular bioactivity. In addition, more and more electron clouds were caused by steric hindrance, which restricts the passage of electrons to the electrode surface, resulting in the resistance increase of the chemiresistive sensors. Therefore, the resistance change of the system can be directly used to indicate the concentration of E. coli in the sample solution. In order to determine the kinetics of bacteria-antibody binding event, the MG-based chemiresistive sensor was made in contact with 107 cfu ml-1 of E. coli bacteria solution. As shown in Fig. 4, the graphene resistance was measured in every 5 mins from 0 to 50 mins, which shows a trend of resistance increase in time. This is attributed to the gradual increase in the number of E. coli captured by the antibodies on the graphene surface. The resistance values increased sharply and reached a plateau as the incubation time increases. These results suggest that the binding reaction finished in about 10-15 mins due to the saturation of bacteria-antibody binding. In order to avoid the exhaustion of antibodies for sensing purpose, we decide to choose 5 mins for the time lag in all the following sensing experiments. 9
Figure 4. Recording of the resistance change of anti-E. coli antibody functionalized MG-based chemiresistive sensors when interacting with E. coli solution (incubated with 107 cfu ml-1 E. coli for 50 mins). The resistance spectra was obtained in Fig. 5(a) for the MG-based chemiresistive sensor when bacteria solution was injected into the microchannel and let sit for 5 mins. The tested concentrations of the E. coli K12 solutions ranged from as low as 2.4х5 to 2.4х57 cfu ml-1. In Fig. 5, ∆R is defined as the absolute change of resistance
(1) where R0 is the initial resistance of the non-bacteria chemiresistive sensor and Rx is the actual resistance value of the chemiresistor after 5 mins of injection and sitting of E. coli K12 samples with various concentrations of 2.4х5X cfu ml-1 (x = 0 to 8). The result shows that the resistance of the MG-based chemiresistive sensors tend to increase with logarithmically increasing adsorption of E. coli on the sensor surface. The (∆R) was finally plateaued at about 260 ohms, which means that the antibody was probably completely consumed by E. coli in 5 min at high concentrations. These results confirm that the combination of MG-based chemiresistive sensor with the real-time measurement of the resistance change is appropriate for the development of highly sensitive pathogen bacteria detection sensors for a wide range of concentration. We also explored another sampling modes, where the sample flow rate was maintained at 2μl min-1 after a series of E. coli K12 solutions with different concentrations were injected in the microchannel. The results are shown in Fig. 5(b), and the increasing trend is very similar to the injection-and-stop sample method. This means that the MG-based chemiresistive biosensor is suitable for being integrated into continuous-flow microfluidic systems for more sophisticated sensing and diagnostics tasks. (a)
10
(b)
Figure 5. (a) Resistance spectra of anti-E. coli antibody functionalized MG-based chemiresistive sensor injected with different concentrations of E. coli solution (every concentration solution was injected and sit in the microchannel for 5 mins). (b) Resistance spectra of anti-E. coli antibody functionalized MG-based chemiresistive sensor injected with a constant flow (2μl min-1) of different concentrations of E. coli K12 solutions (every concentration solution was injected and maintained at the constant flowrate for 5 mins).
3. Conclusions In this work, we have developed a novel microfluidic approach to detect E. coli K12 using a monolayer graphene chemiresistive biosensor. The microchannel was fabricated using PDMS, and Au nanoparticles and streptavidin combination on the graphene were applied to conjugate the antibody. The solution samples were injected into the microchannel for testing, which was achieved by monitoring the graphene resistance changes during the adsorption of E. coli by the 11
antibody. A linear increasing trend of ∆R was observed with logarithmically increasing E. coli concentrations from 2.4х5 to 2.4х56 cfu ml-1. Both injection-and-stop and constant-injection modes of the samples were tested and the results indicate a similar trend. Overall, the developed microfluidic sensor demonstrates rapid and sensitive bacterial detection. One major limitation here is that the graphene sensors cannot be recycled for reuse. Therefore, these sensors will have to be disposable in real-life applications. But the portability, low-cost, and ease of fabrication of the sensor provides a promising approach for future development of multiplexed microfluidic biosensor platforms for a wide variety of applications.
Conflicts of interest The authors declare no conflict of interest.
Acknowledgments This work was supported by an Early Career Faculty grant (80NSSC17K0522) from NASA's Space Technology Research Grants Program.
12
References [1] J.B. Kaper, J.P. Nataro, H.L. Mobley, Pathogenic escherichia coli, Nature reviews microbiology, 2(2004) 123. [2] A.M. Svennerholm, From cholera to enterotoxigenic Escherichia coli (ETEC) vaccine development, Indian J Med Res, 133(2011) 188-96. [3] T.H. Pennington, E. coli O157 outbreaks in the United Kingdom: past, present, and future, Infect Drug Resist, 7(2014) 211-22. [4] E. Velliou, E. Noriega, E. Van Derlinden, L. Mertens, K. Boons, A. Geeraerd, et al., The effect of colony formation on the heat inactivation dynamics of Escherichia coli K12 and Salmonella Typhimurium, Food research international, 54(2013) 1746-52. [5] P. Arora, A. Sindhu, N. Dilbaghi, A. Chaudhury, Biosensors as innovative tools for the detection of food borne pathogens, Biosensors and Bioelectronics, 28(2011) 1-12. [6] C. Burtscher, S. Wuertz, Evaluation of the use of PCR and reverse transcriptase PCR for detection of pathogenic bacteria in biosolids from anaerobic digestors and aerobic composters, Appl Environ Microbiol, 69(2003) 4618-27. [7] H. Beckers, P. Tips, P. Soentoro, E. Delfgou-Van Asch, R. Peters, The efficacy of enzyme immunoassays for the detection of salmonellas, Food Microbiology, 5(1988) 147-56. [8] N.J. Ronkainen, H.B. Halsall, W.R.J.C.S.R. Heineman, Electrochemical biosensors, 39(2010) 1747-63. [9] L. Sepunaru, K. Tschulik, C. Batchelor-McAuley, R. Gavish, R.G. Compton, Electrochemical detection of single E. coli bacteria labeled with silver nanoparticles, Biomaterials science, 3(2015) 816-20. [10] Y. Wen, F.Y. Li, X. Dong, J. Zhang, Q. Xiong, P.J.A.h.m. Chen, The Electrical Detection of Lead Ions Using Gold‐Nanoparticle‐and DNAzyme‐Functionalized Graphene Device, 2(2013) 271-4. [11] K. Bhalla, M. Shotten, A. Cohen, M. Brauer, S. Shahraz, R. Burnett, et al., Transport for health: the global burden of disease from motorized road transport2014. [12] W. Wu, S. Zhao, Y. Mao, Z. Fang, X. Lu, L. Zeng, A sensitive lateral flow biosensor for Escherichia coli O157: H7 detection based on aptamer mediated strand displacement amplification, Analytica chimica acta, 861(2015) 62-8. [13] K. Glynou, P.C. Ioannou, T.K. Christopoulos, V.J.A.C. Syriopoulou, Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization, 75(2003) 4155-60. [14] Y. Guo, Y. Wang, S. Liu, J. Yu, H. Wang, M. Cui, et al., Electrochemical immunosensor assay (EIA) for sensitive detection of E. coli O157: H7 with signal amplification on a SG–PEDOT–AuNPs electrode interface, Analyst, 140(2015) 551-9. [15] C. Lee, X. Wei, J.W. Kysar, J. Hone, Measurement of the elastic properties and intrinsic strength of monolayer graphene, science, 321(2008) 385-8. [16] J.W. Suk, R.D. Piner, J. An, R.S.J.A.n. Ruoff, Mechanical properties of monolayer graphene oxide, 4(2010) 6557-64. [17] C. Chen, S. Rosenblatt, K.I. Bolotin, W. Kalb, P. Kim, I. Kymissis, et al., Performance of monolayer graphene nanomechanical resonators with electrical readout, 4(2009) 861. [18] Y. Wang, J. Ping, Z. Ye, J. Wu, Y. Ying, Impedimetric immunosensor based on gold nanoparticles modified graphene paper for label-free detection of Escherichia coli O157: H7, Biosensors and Bioelectronics, 49(2013) 492-8. 13
[19] K. Krishnamoorthy, M. Veerapandian, L.-H. Zhang, K. Yun, S.J. Kim, Antibacterial efficiency of graphene nanosheets against pathogenic bacteria via lipid peroxidation, The journal of physical chemistry C, 116(2012) 17280-7. [20] J. Zhao, A. Hashmi, J. Xu, W. Xue, A compact lab-on-a-chip nanosensor for glycerol detection, Applied Physics Letters, 100(2012) 243109. [21] A.M. Clark, K.M. Sousa, C. Jennings, O.A. MacDougald, R.T. Kennedy, Continuous-flow enzyme assay on a microfluidic chip for monitoring glycerol secretion from cultured adipocytes, Analytical chemistry, 81(2009) 2350-6. [22] S. Viswanathan, T.N. Narayanan, K. Aran, K.D. Fink, J. Paredes, P.M. Ajayan, et al., Graphene–protein field effect biosensors: glucose sensing, Materials Today, 18(2015) 513-22. [23] Y. Xia, G.M. Whitesides, Soft lithography, Annual review of materials science, 28(1998) 153-84. [24] P. Kim, K.W. Kwon, M.C. Park, S.H. Lee, S.M. Kim, K.Y. Suh, Soft lithography for microfluidics: a review, (2008). [25] Y.Y. Wang, Z.H. Ni, T. Yu, Z.X. Shen, H.M. Wang, Y.H. Wu, et al., Raman studies of monolayer graphene: the substrate effect, The Journal of Physical Chemistry C, 112(2008) 10637-40. [26] A. Das, B. Chakraborty, A. Sood, Raman spectroscopy of graphene on different substrates and influence of defects, Bulletin of Materials Science, 31(2008) 579-84. [27] F. Tausig, F.J. Wolf, Streptavidin-a substance with avidin-like properties produced by microorganisms, Biochemical and biophysical research communications, 14(1964) 205-9. [28] A. Jain, K. Cheng, The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis, Journal of controlled release, 245(2017) 27-40. [29] T. Huberman, Y. Eisenberg-Domovich, G. Gitlin, T. Kulik, E.A. Bayer, M. Wilchek, et al., Chicken Avidin Exhibits Pseudo-catalytic Properties BIOCHEMICAL, STRUCTURAL, AND ELECTROSTATIC CONSEQUENCES, Journal of Biological Chemistry, 276(2001) 32031-9. [30] R.K. DeLong, C.M. Reynolds, Y. Malcolm, A. Schaeffer, T. Severs, A. Wanekaya, Functionalized gold nanoparticles for the binding, stabilization, and delivery of therapeutic DNA, RNA, and other biological macromolecules, Nanotechnology, science and applications, 3(2010) 53. [31] L. Yang, Y. Li, G.F. Erf, Interdigitated array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia c oli O157: H7, Analytical chemistry, 76(2004) 1107-13.
14
Monolayer graphene chemiresistive biosensor for rapid bacteria detection in a microchannel Weiqi Zhao , Yunyi Xing , Yang Lin , Yuan Gao , Mengren Wu , Jie Xu PII: DOI: Reference:
S2666-0539(20)30001-1 https://doi.org/10.1016/j.snr.2020.100004 SNR 100004
To appear in:
Sensors and Actuators Reports
Received date: Revised date: Accepted date:
21 December 2019 4 February 2020 12 February 2020
Please cite this article as: Weiqi Zhao , Yunyi Xing , Yang Lin , Yuan Gao , Mengren Wu , Jie Xu , Monolayer graphene chemiresistive biosensor for rapid bacteria detection in a microchannel, Sensors and Actuators Reports (2020), doi: https://doi.org/10.1016/j.snr.2020.100004
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Highlights
Microfluidic chemiresistive biosensors are fabricated using monolayer graphene, gold nanoparticles, streptavidin-antibody system, and PDMS.
The biosensor captures E. coli bacteria and performs sensing via gate-free resistance response.
The developed biosensor is proved to be simple, rapid and sensitive for bacteria detection.
1
Monolayer graphene chemiresistive biosensor for rapid bacteria detection in a microchannel
Weiqi Zhao, Yunyi Xing, Yang Lin, Yuan Gao, Mengren Wu and Jie Xu* Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA E-mail: [email protected]
Graphical abstract
2
Abstract Bacteria, such as Escherichia coli (E. coli), can cause food poisoning and serious diseases. In fact, E. coli has emerged multiple times as a severe public health concern in recent years. Therefore, it is important to detect bacteria like E. coli in simple, rapid and sensitive approaches. In this study, a gate-free chemiresistive biosensor based on monolayer graphene (MG) is proposed to detect E. coli, thanks to the high sensitivity stemming from the anti-E. coli antibody-coated graphene. The immobilization of the antibodies is performed via streptavidin and biotin conjugation on the graphene surface. Compared to conventional bacterial biosensors, the proposed chemiresistive biosensor captures the E. coli bacteria on the surface of the sensor and performs the detection through electric readouts, other than optical signals. That is the resistance measured in the biosensor increases along with the increase of concentration of the bacteria. To further extend the capabilities of this biosensor and provide a controllable test flow, a microchannel is integrated on the graphene surface. The results show that the developed chemiresistive biosensor is able to detect E. coli in trace concentration down to 12 cfu ml-1, indicating an excellent sensing performance. The proposed monolayer graphene based chemiresistive biosensor clearly manifest its advantages of low-cost, ease of fabrication, portability and good sensitivity, as well as the potential for rapid in-situ bacterial detection. Keywords – Bacteria, Escherichia coli (E. coli), Monolayer graphene (MG), Chemiresistive Biosensor, Concentration, Microchannel
3
Introduction Bacteria accompany our lives, but sometimes they will cause problems in our health. For example, Escherichia coli (E. coli), which usually colonizes human intestines right after birth [1], inevitably affects human beings throughout their entire life spans. The discovery of E. coli can be dated back to 1885 by Theodor Escherich, and it was originally considered as a healthy mutualism bacterium helping human beings with synthesis of vitamin K2 [2]. In 1996, scientists found that E. coli can also cause food poisoning in human beings, as evidenced by a worldwide outbreak of food poisoning occurred in Scotland [3]. In addition, E. coli infections can cause diarrhea and lead to life-threatening conditions, such as kidney failure, high blood pressure, chronic kidney disease, and neurologic problems. To minimize the impact of E. coli on human society, efficient diagnosis of the bacteria is of utmost importance. At present, a variety of methods have been developed to detect and determine the concentration of E. coli. For instance, one of the most straightforward methods is through cell culture, in which E. coli is cultured on a petri dish for 24 h at 37 °C in an incubator. By means of counting the number of E. coli colonies, the concentration of E. coli can be determined [4]. Nonetheless, this method usually requires long preparation time. Therefore it has been gradually substituted by other approaches. For example, nucleic acid-based polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) technology could give rise to a much more accurate result in a shorter time [5-7]. Nevertheless, they are not satisfactory for rapid detection as they are costly, time consuming and require immobile laboratory settings. Recently, various electrochemical based biosensors for the detection of E. coli pathogen has been developed [8]. For example, anodic particle coulometry technique has been combined with cyclic voltammetry (CV), and the concentration of the E. coli decreases as the absorption spectra mean decreases [9]. Moreover, nanomaterials such as gold nanorods and gold nanoparticles (AuNPs) have also been incorporated into sensing assays to further enhance the specificity and sensitivity of detection [10]. For example, Bhalla and coworkers have shown that the gold nanoparticles can increase the sensitivity for biosensor significantly. Specifically, the gold nanoparticles helped the detection of cardiac troponin I (cTnI) at a concentration as low as 0.2 ng mL−1, which is much better than ELISA tests of 4.3 ng mL−1 [11]. Depending on the function of the gold nanoparticles, it can be linked to aptamer for capturing target bacteria. In order to find the adaptor on the E. coli membrane for aptamer, the amplified single-strand DNA need to be translated based on the DNA array of E. coli, in which the concentration of E. coli can be obtained [12]. However, the DNA screening of aptamer is relatively difficult, and the specificity provided by aptamers is often limited. In this study a chemiresistor biosensor based on monolayer graphene is developed with electrical readouts for E. coli detection. Instead of using aptamers, we choose to use antibody-streptavidin system together with AuNPs for high specificity and high sensitivity bacteria recognition. Streptavidin has been proved to be effective when it is linked to gold nanoparticles for biosensing [13]. The sensing element in our system is monolayer graphene (MG). Graphene is widely used in biosensors because of its special electrical properties [14]. Graphene can transfer 15000 cm2 Vs-1 electrons at room temperature while the resistance is only around 10-6 ohm.cm. Moreover, multiple studies of 4
monolayer-graphene have confirmed their enormous stiffness and ultrathin thickness [15-17]. Thereby monolayer graphene with large contact area is naturally suitable for providing abundant binding sites for sensing purpose. Graphene’s electric properties, such as conductivity, is very sensitive to its environment, not to mention chemical bindings on its surface. Hence, we immobilize integrated AuNPs and streptavidin-antibody system on the graphene surface for E. coli capturing and monitor the electrical signals generated. Gold nanoparticles were spread over monolayer graphene to produce conductivity, meanwhile, the electrode was produced from gold nanoparticle which can increase the sensitivity of biosensors [18]. As observed in the experiments, when the system binds with E. coli, the resistance of the monolayer graphene is increased. Therefore, the bio-signals of E. coli binding are converted into electrical signals, and the concentration of E. coli is then verified. It is worth mentioning that both graphene and gold also have a special function in antibacterial, which makes the device safer to operate for pathogen-related applications [19]. With the development of microfluidic technology, different types of lab-on-a-chip devices with in-channel chemical sensors have been developed. For instance, the microfluidic system based on single-walled carbon nanotubes [20] and based on enzyme assays [21] have been integrated for detecting glycerol concentration. In addition, a microchannel system also can be used in the glucose detection [22]. Research activities and advances in lab-on-a-chip devices have revolutionized the field of biosensing research, since sensing in a microfluidic environment often benefits from stable and precise sample control, parallel and multiplexed assay capabilities, and scaling-up potential. By nature, electrochemical sensors are easily and effectively to integrate with microfluidic platforms because of their simple structure. In this paper, the developed biosensor is integrated into a lab-on-a-chip device for the detection of different concentration of E. coli solution.
1. Material and methods 1.1. Materials Monolayer graphene coated on a square substrate of Si/SiO₂ was purchased from Graphenea, Inc. Gold/palladium nano-particle target was purchased from Ted Pella, Inc. Phosphate buffered saline (PBS, 0.1 mol l-1, pH 7.4) with 0.1% Tween 20 was purchased from Sigma Aldrich Inc. Streptavidin was purchased from AnaSpec, Inc. E. coli K12 ER2925 were purchased from New England Biolab, Inc. Biotinylated anti-E. coli (from rabbit) antibody was purchased from abcam, Inc. Goat anti-rabbit IgG (Heavy & Light Chain) antibody (Atto 488) was purchased from Antibody-Online, Inc. The tryptic soy broth and tryptic soy agar for medium were purchased from Thermo Fisher Scientific, Inc. 1.2. Device fabrication The overall detection system is illustrated in Fig. 1(a). Monolayer-graphene substrate with a size of 1 cm by 1 cm was first scrutinized to ensure a good quality. Afterwards, two electrodes were coated on the surface of graphene substrate with gold/palladium alloy target (99.99% Au:Pd, 60:40 ratio) supported by a low pressure argon gas chamber (0.05 mbar) using a Au/Pd Polaron 5
sputter coater E5100 series II. As shown in Fig. 1(b), a predesigned mask was fixed on the upper surface of the substrate, after which the sputter coater was used to deposit two 15 nm thick Au/Pd continuous films as electrodes (for 10 minutes). After stripping the mask, the sputtering coater was used for a second time, but the second time coating was on the full surface of the graphene substrate and was only for 10 seconds. This way, the center of graphene substrate had been coated with nanoparticle stratum granulosum as Au particle linker. After the preparation of the electrodes, two PDMS layers were added onto the substrate to create a microchannel. The first layer was the microchannel layer of PDMS which was used to cover the top of graphene piece. The width, length, and the depth of the microchannel are 800μm, 8mm and 1mm, respectively. The dimensions are chosen because they can be made conveniently using low-cost technology. Specifically, the PDMS layers were created via standard soft lithography [23, 24]. Specifically, the mold of the microchannel was fabricated on a polymethyl methacrylate (PMMA) plate by a micro-milling machine (CNC Mini-Mill/3 PRO). The PDMS pre-mixture was then mixed, degassed, and poured onto the mold, and kept in an oven for 8 min at 75℃. While the PDMS was still a little bit sticky, it was adhered onto the graphene, and punched the injection and extraction holes on the two sides of the channel. After that, the graphene was placed with the PDMS layer down on the plate. Then the second layer PDMS liquid was poured onto the substrate and sealed the graphene substrate into the PDMS. The whole device was placed in the oven for 20 mins at 75 ℃. The two grooves were cut on the two sides of the electrodes, which were used to gain electric contact on the electrodes for electric measurements (Fig. 2). Finally, the microchannel was rinsed with deionized water (DI water), in order to remove impurities in the microchannel. Afterwards, the microchannel was injected and incubated overnight with 15µl streptavidin (1mg ml-1) at 4 ℃. After rinsing with buffer (PBS containing 0.005% Tween 20) and DI water thoroughly, the graphene was treated by 15 μl of 0.5 mg ml-1 biotinylated anti-E. coli antibodies for 2 hours, followed by rinsing with DI water. After the fabrication of devices, they can be used immediately for the detection of bacteria. In our experiment, a series of E. coli K12 solutions with different concentrations were injected into the microchannel for 5 mins consecutively for E. coli detection and measurements. If the devices are not to be used immediately, it is recommended to seal an antibody stabilizer PBS buffer in the microchannels for preserving the proteins and antibodies for a relatively long time, and the devices should be kept in a refrigerator (2-8 ℃). 1.3. Bacteria preparation The original E. coli K12 was cultured in the tryptic soy broth solution and incubated in the incubator for 16 hours at 37 ℃ with 246 RPM stir speed. The bacterial population were maximized, and then the initial concentrations of bacteria samples were obtained by serial dilution. The tryptic soy agar was used in a petri dish to culture E. coli, in order to verify the initial count of the E. coli, using the so-called plate count method.
6
Figure 1. (a) The structure of the PDMS microchannel. The PDMS microchannel was fixed on the monolayer-graphene with electrodes. Under the PDMS microchannel is the structure of the biosensor in the center of the microchannel. (b) The sputter coater was used to coat two sides of electrodes. The mask was covered on the upper surface of the graphene. 1.4.Electrical measurement Electrical measurements were performed on monolayer-graphene biosensor using a micropositioner with a Keithley 4200 source meter system at room temperature. The sensing signal of the device was recorded by LabView software monitoring the change between two electrodes in the current (I) for a given source voltage (U) from 0 V to 2 V when the device was exposed to different concentrations of E. coli solution. The scanning electron microscopy (SEM) images were obtained using a JSM 6320F SEM. Raman spectra was obtained on a micro-Raman spectrometer excited by 532 nm laser excitation. Fig. 2 shows the actual graphene biosensor 7
integrated in a PDMS microchannel. Two grooves on the two sides of the electrodes were used to get access to the electrodes.
Figure 2. A photo of the proposed microfluidic chemiresistive biosensor.
2. Results and discussion 2.1. Characterization of monolayer-graphene The SEM image of the MG is depicted in Fig. 3(a), which shows an area in between the two electrodes of the sensor. As observed, the graphene film is continuous, uniform, and dominantly single-layered structure across the entire surface. Raman spectra measurements were performed to further confirm the presence of the high-quality MG layer. As shown in Fig. 3(b), the MG displays at two prominent peaks at 1580 cm-1 and 2676 cm-1, corresponding to the well-documented G and 2D, respectively [25, 26]. The fact that the 2D peak is greater than the G peak indicates the presence of a defect-free MG layer on the chemiresistor sensor, which is also in accordance with the evidence from the SEM image (Fig. 3(a)).
(a)
(b)
Figure 3. (a) A small area of the MG-based chemiresistive sensor in between the Au/Pd electrodes was characterized by SEM; (b) Raman spectra of the MG chemiresistive sensor. 8
2.2. Characterization of linker The linkers were applied on the graphene substrate to capture the E. coli, which then causes a change in resistance in MG for detection. The primary linkers are Au nanoparticle, Streptavidin and antibody on the graphene. Streptavidin is the most widely used analogue of avidin [27]. The avidin-biotin affinity interaction was approximately 103 to 106 times higher than an antibody-antigen interaction [28]. Therefore, application of streptavidin in this biosensor enables very stable linking (compared to antibody-antigen). Because streptavidin can protect the biotinyl esters from hydrolysis, but avidin can augment this hydrolysis [29]. Au nanoparticles and streptavidin are biologically specific binding. These functional groups of water-soluble nanoparticles are usually carboxylic acids, which stabilize nanoparticles by electrostatic repulsion and can be used to conjugate other molecules to particles [30]. In order to verify these linkers (Au nanoparticle, streptavidin and anti-E. coli antibody) can be linked together very well, we used labelled secondary antibody to verify that the anti-E. coli had fixed on the surface. The Au nanoparticles were coated on a glass slide. Meanwhile a circle was drawn on the glass slide, and the streptavidin and anti-E. coli solution were dropped on this area respectively. After that, PBS solution was applied to rinse the slide surface three times. The secondary antibody with fluorescence under the laser microscope was observed in the circled area only. Therefore, it was confirmed that these linkers had been jointed together and the other linkers which were not linked had been rinsed out. 2.3. Results and discussion In the bacteria-antibody binding system, the amount of the bacteria-antibodies bond being produced during the sensing process directly affects the resistance of the MG. It is reported that the membrane of nature biological cells shows a resistance of 102-105 Ω cm-1 [18, 31]. When the bacteria cells attach to the surface of graphene film, the formation of bacteria-antibody conjugation could produce kinetics barrier for the electron transfer process, which may induce more distinct changes in the carrier hole density in graphene film through polarization of cell-surface charges, intracellular bioactivity. In addition, more and more electron clouds were caused by steric hindrance, which restricts the passage of electrons to the electrode surface, resulting in the resistance increase of the chemiresistive sensors. Therefore, the resistance change of the system can be directly used to indicate the concentration of E. coli in the sample solution. In order to determine the kinetics of bacteria-antibody binding event, the MG-based chemiresistive sensor was made in contact with 107 cfu ml-1 of E. coli bacteria solution. As shown in Fig. 4, the graphene resistance was measured in every 5 mins from 0 to 50 mins, which shows a trend of resistance increase in time. This is attributed to the gradual increase in the number of E. coli captured by the antibodies on the graphene surface. The resistance values increased sharply and reached a plateau as the incubation time increases. These results suggest that the binding reaction finished in about 10-15 mins due to the saturation of bacteria-antibody binding. In order to avoid the exhaustion of antibodies for sensing purpose, we decide to choose 5 mins for the time lag in all the following sensing experiments. 9
Figure 4. Recording of the resistance change of anti-E. coli antibody functionalized MG-based chemiresistive sensors when interacting with E. coli solution (incubated with 107 cfu ml-1 E. coli for 50 mins). The resistance spectra was obtained in Fig. 5(a) for the MG-based chemiresistive sensor when bacteria solution was injected into the microchannel and let sit for 5 mins. The tested concentrations of the E. coli K12 solutions ranged from as low as 2.4х5 to 2.4х57 cfu ml-1. In Fig. 5, ∆R is defined as the absolute change of resistance
(1) where R0 is the initial resistance of the non-bacteria chemiresistive sensor and Rx is the actual resistance value of the chemiresistor after 5 mins of injection and sitting of E. coli K12 samples with various concentrations of 2.4х5X cfu ml-1 (x = 0 to 8). The result shows that the resistance of the MG-based chemiresistive sensors tend to increase with logarithmically increasing adsorption of E. coli on the sensor surface. The (∆R) was finally plateaued at about 260 ohms, which means that the antibody was probably completely consumed by E. coli in 5 min at high concentrations. These results confirm that the combination of MG-based chemiresistive sensor with the real-time measurement of the resistance change is appropriate for the development of highly sensitive pathogen bacteria detection sensors for a wide range of concentration. We also explored another sampling modes, where the sample flow rate was maintained at 2μl min-1 after a series of E. coli K12 solutions with different concentrations were injected in the microchannel. The results are shown in Fig. 5(b), and the increasing trend is very similar to the injection-and-stop sample method. This means that the MG-based chemiresistive biosensor is suitable for being integrated into continuous-flow microfluidic systems for more sophisticated sensing and diagnostics tasks. (a)
10
(b)
Figure 5. (a) Resistance spectra of anti-E. coli antibody functionalized MG-based chemiresistive sensor injected with different concentrations of E. coli solution (every concentration solution was injected and sit in the microchannel for 5 mins). (b) Resistance spectra of anti-E. coli antibody functionalized MG-based chemiresistive sensor injected with a constant flow (2μl min-1) of different concentrations of E. coli K12 solutions (every concentration solution was injected and maintained at the constant flowrate for 5 mins).
3. Conclusions In this work, we have developed a novel microfluidic approach to detect E. coli K12 using a monolayer graphene chemiresistive biosensor. The microchannel was fabricated using PDMS, and Au nanoparticles and streptavidin combination on the graphene were applied to conjugate the antibody. The solution samples were injected into the microchannel for testing, which was achieved by monitoring the graphene resistance changes during the adsorption of E. coli by the 11
antibody. A linear increasing trend of ∆R was observed with logarithmically increasing E. coli concentrations from 2.4х5 to 2.4х56 cfu ml-1. Both injection-and-stop and constant-injection modes of the samples were tested and the results indicate a similar trend. Overall, the developed microfluidic sensor demonstrates rapid and sensitive bacterial detection. One major limitation here is that the graphene sensors cannot be recycled for reuse. Therefore, these sensors will have to be disposable in real-life applications. But the portability, low-cost, and ease of fabrication of the sensor provides a promising approach for future development of multiplexed microfluidic biosensor platforms for a wide variety of applications.
Conflicts of interest The authors declare no conflict of interest.
Acknowledgments This work was supported by an Early Career Faculty grant (80NSSC17K0522) from NASA's Space Technology Research Grants Program.
12
References [1] J.B. Kaper, J.P. Nataro, H.L. Mobley, Pathogenic escherichia coli, Nature reviews microbiology, 2(2004) 123. [2] A.M. Svennerholm, From cholera to enterotoxigenic Escherichia coli (ETEC) vaccine development, Indian J Med Res, 133(2011) 188-96. [3] T.H. Pennington, E. coli O157 outbreaks in the United Kingdom: past, present, and future, Infect Drug Resist, 7(2014) 211-22. [4] E. Velliou, E. Noriega, E. Van Derlinden, L. Mertens, K. Boons, A. Geeraerd, et al., The effect of colony formation on the heat inactivation dynamics of Escherichia coli K12 and Salmonella Typhimurium, Food research international, 54(2013) 1746-52. [5] P. Arora, A. Sindhu, N. Dilbaghi, A. Chaudhury, Biosensors as innovative tools for the detection of food borne pathogens, Biosensors and Bioelectronics, 28(2011) 1-12. [6] C. Burtscher, S. Wuertz, Evaluation of the use of PCR and reverse transcriptase PCR for detection of pathogenic bacteria in biosolids from anaerobic digestors and aerobic composters, Appl Environ Microbiol, 69(2003) 4618-27. [7] H. Beckers, P. Tips, P. Soentoro, E. Delfgou-Van Asch, R. Peters, The efficacy of enzyme immunoassays for the detection of salmonellas, Food Microbiology, 5(1988) 147-56. [8] N.J. Ronkainen, H.B. Halsall, W.R.J.C.S.R. Heineman, Electrochemical biosensors, 39(2010) 1747-63. [9] L. Sepunaru, K. Tschulik, C. Batchelor-McAuley, R. Gavish, R.G. Compton, Electrochemical detection of single E. coli bacteria labeled with silver nanoparticles, Biomaterials science, 3(2015) 816-20. [10] Y. Wen, F.Y. Li, X. Dong, J. Zhang, Q. Xiong, P.J.A.h.m. Chen, The Electrical Detection of Lead Ions Using Gold‐Nanoparticle‐and DNAzyme‐Functionalized Graphene Device, 2(2013) 271-4. [11] K. Bhalla, M. Shotten, A. Cohen, M. Brauer, S. Shahraz, R. Burnett, et al., Transport for health: the global burden of disease from motorized road transport2014. [12] W. Wu, S. Zhao, Y. Mao, Z. Fang, X. Lu, L. Zeng, A sensitive lateral flow biosensor for Escherichia coli O157: H7 detection based on aptamer mediated strand displacement amplification, Analytica chimica acta, 861(2015) 62-8. [13] K. Glynou, P.C. Ioannou, T.K. Christopoulos, V.J.A.C. Syriopoulou, Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization, 75(2003) 4155-60. [14] Y. Guo, Y. Wang, S. Liu, J. Yu, H. Wang, M. Cui, et al., Electrochemical immunosensor assay (EIA) for sensitive detection of E. coli O157: H7 with signal amplification on a SG–PEDOT–AuNPs electrode interface, Analyst, 140(2015) 551-9. [15] C. Lee, X. Wei, J.W. Kysar, J. Hone, Measurement of the elastic properties and intrinsic strength of monolayer graphene, science, 321(2008) 385-8. [16] J.W. Suk, R.D. Piner, J. An, R.S.J.A.n. Ruoff, Mechanical properties of monolayer graphene oxide, 4(2010) 6557-64. [17] C. Chen, S. Rosenblatt, K.I. Bolotin, W. Kalb, P. Kim, I. Kymissis, et al., Performance of monolayer graphene nanomechanical resonators with electrical readout, 4(2009) 861. [18] Y. Wang, J. Ping, Z. Ye, J. Wu, Y. Ying, Impedimetric immunosensor based on gold nanoparticles modified graphene paper for label-free detection of Escherichia coli O157: H7, Biosensors and Bioelectronics, 49(2013) 492-8. 13
[19] K. Krishnamoorthy, M. Veerapandian, L.-H. Zhang, K. Yun, S.J. Kim, Antibacterial efficiency of graphene nanosheets against pathogenic bacteria via lipid peroxidation, The journal of physical chemistry C, 116(2012) 17280-7. [20] J. Zhao, A. Hashmi, J. Xu, W. Xue, A compact lab-on-a-chip nanosensor for glycerol detection, Applied Physics Letters, 100(2012) 243109. [21] A.M. Clark, K.M. Sousa, C. Jennings, O.A. MacDougald, R.T. Kennedy, Continuous-flow enzyme assay on a microfluidic chip for monitoring glycerol secretion from cultured adipocytes, Analytical chemistry, 81(2009) 2350-6. [22] S. Viswanathan, T.N. Narayanan, K. Aran, K.D. Fink, J. Paredes, P.M. Ajayan, et al., Graphene–protein field effect biosensors: glucose sensing, Materials Today, 18(2015) 513-22. [23] Y. Xia, G.M. Whitesides, Soft lithography, Annual review of materials science, 28(1998) 153-84. [24] P. Kim, K.W. Kwon, M.C. Park, S.H. Lee, S.M. Kim, K.Y. Suh, Soft lithography for microfluidics: a review, (2008). [25] Y.Y. Wang, Z.H. Ni, T. Yu, Z.X. Shen, H.M. Wang, Y.H. Wu, et al., Raman studies of monolayer graphene: the substrate effect, The Journal of Physical Chemistry C, 112(2008) 10637-40. [26] A. Das, B. Chakraborty, A. Sood, Raman spectroscopy of graphene on different substrates and influence of defects, Bulletin of Materials Science, 31(2008) 579-84. [27] F. Tausig, F.J. Wolf, Streptavidin-a substance with avidin-like properties produced by microorganisms, Biochemical and biophysical research communications, 14(1964) 205-9. [28] A. Jain, K. Cheng, The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis, Journal of controlled release, 245(2017) 27-40. [29] T. Huberman, Y. Eisenberg-Domovich, G. Gitlin, T. Kulik, E.A. Bayer, M. Wilchek, et al., Chicken Avidin Exhibits Pseudo-catalytic Properties BIOCHEMICAL, STRUCTURAL, AND ELECTROSTATIC CONSEQUENCES, Journal of Biological Chemistry, 276(2001) 32031-9. [30] R.K. DeLong, C.M. Reynolds, Y. Malcolm, A. Schaeffer, T. Severs, A. Wanekaya, Functionalized gold nanoparticles for the binding, stabilization, and delivery of therapeutic DNA, RNA, and other biological macromolecules, Nanotechnology, science and applications, 3(2010) 53. [31] L. Yang, Y. Li, G.F. Erf, Interdigitated array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia c oli O157: H7, Analytical chemistry, 76(2004) 1107-13.
14