Self-Assembling of Electrochemical Glucose Biosensor with Bacteriostatic Materials via Layer-by-Layer Method

Poly-diallyl-dimethyl-ammonium chloride (PDDA) solution was used to disperse carbon nanotubes (CNTs) to form a stable PDDA- CNTs aqueous dispersion. The negatively charged glucose oxidase (GOx) and positively charged PDDA-CNTs composite were used to prepare multilayer biosensing ﬁlms on glassy carbon electrodes (GCE) via layer-by-layer (LBL) self-assembly technique. The optimum number of layers on GCE was 4. A mixture of 3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate (DMAPS) and Graphene (GR) was dropped on the multilayer ﬁlms to prepare a bacteriostatic glucose biosensor. The results show that CNTs could evenly disperse in the PDDA ﬁlms and the multilayer PDDA-CNTs ﬁlms can signiﬁcantly improve the catalytic current response toward glucose (Glu). The biosensor could detect glucose linearly from 16.5 to 214.3 mM. The bacteriostatic properties of the sensor were ensured by the bacteriostatic characteristic of DMAPS.

Antimicrobial or bacteriostatic material has been widely used in industries such as textile, food fermentation and medical device industry. 18,19 In the process of bio-fermentation, the detection of various biochemical parameters (biomass, cell activity, substrate, nutrition, products and metabolites) forms the basis of controlling process of the fermentation. 20 The growth rate of micro-organism can be monitored by the consumption of glucose. Consequently, the abnormal phenomenon of fermentation process can be forecasted by the timely detection of glucose. 21 However, bacterium can be easily adsorbed on the surface of the glucose sensors to form a bio-film. The bio-film can block the substrate close to the electrode, and the accuracy of the detection can be affected. 22 In these cases, it is especially necessary to use antimicrobial or bacteriostatic glucose biosensors. But there are very few studies reporting the antimicrobial or bacteriostatic electrochemical glucose biosensor. In this paper, a bacteriostatic film is built on GCE via layer-by-layer (LbL) self-assembly method and used to construct a glucose sensor.
It is generally known that chemical compounds contain quaternized ammonium groups and that zwitterionic sulfopropylbetaine shows bacteriostatic or antimicrobial properties, 23 besides the zwitterionic materials show outstanding protein resistance performance. 24,25 A typical sulfobetaine, 3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate (DMAPS) (Scheme 1), which contains a sulfonate group and a quarternized ammonium separated by an alkyl spacer, has outstanding antimicrobial properties.
Layer-by-layer (LbL) self-assembly method is a useful and versatile technic to fabricate functional molecular assemblies with welldefined architectures. 26,27 Poly(diallyldimethylammonium chloride) (PDDA) is a positively charged polyelectrolyte, and the formation of π bond between PDDA and carbon nanotubes thereby improves the dispersibility of carbon nanotubes in water. [28][29][30] In this paper, we fabricate a glucose sensor with DMAPS, PDDA, CNTs, GOx and Graphene via LBL self-assembling technique.
Electrochemistry measurements were proceeded in 1/15 M phosphate buffer solution (PBS) (pH = 6.98). The buffer solution was made by dissolving 0.04 mol Na 2 HPO 4 and 0.027 mol KH 2 PO 4 in 1.000 L of ultrapure water. Glucose solutions with different concentration were made by dissolving certain amount of anhydrous β-D-glucose in the 1/15 M PBS and were stored at 4 • C (at least 12 hours' store is needed for mutarotation before use). GOX solution (8.0 mg mL −1 ) was prepared by dissolving certain amount of GOX powders in PBS and stored at 4 • C.
Instrumentation.-Ultrapure water was obtained by using Merck Millipore Direct-Q 3.5.8. Electrochemical impedance spectroscopy (EIS), and amperometric measurements such as i-t curves, cyclic voltammetry (CV), were tested by using a CHI 660E (Shanghai Chenhua) electrochemical workstation. A three electrode system which contained a saturated calomel electrode (SCE) as reference electrode and a platinum wire ( 0.5 mm) as counter electrode was used for electrochemical measurements. A pH meter (PHS-3C, Shanghai Leici) is used to measure the pH value, and a conductivity meter (DDS-307, Shanghai Leici) is used to measure the conductivity of the solution.
The effect of DMAPS toward the bacterial growth was observed by using a ultra-violet-visible spectrophotometer (UV-vis) (UV-2550, Shimadzu). The optical density at a wavelength of 600 nm (OD 600 ) was used to characterize the bacteriostasis property of DMAPS. Electrode modification.-Glassy carbon electrode (GCE) was polished with 0.3 and 0.05 μm alumina powder, rinsed thoroughly with water, and sonicated for 2 mins in water and in ethonal, respectively. The cleaned electrode was soaked in the PDDA-CNTs suspension and the GOX solution alternately, each for 30 minutes. The weak adsorption was dissociated by water washing. The modified GCE was dried by nitrogen at the end of each assembly deposition. The last layer is PDDA-CNTs with positive charge ((PDDA-CNTS/GOx) n+0.5 ). Finally, DMAPS-GR was adsorbed into the surface of the PDDA-CNTs, and the above modified electrode modified electrodes immersed in DMAPS-GR dispersion for 30 mins, followed by thoroughly rinsed with water to remove any physically adsorbed components and then dried under nitrogen.

Results and Discussion
The bacteriostasis property of the DMAPS .-PDDA-CNTs are chosen as the anchor layer of the sensor. PDDA is a cationic polymer and is a highly positively charged material because of its amino groups. It can interact with carbon nanotubes by π-π interaction, and improve the dispersibility of carbon nanotubes in water. It can selfassemble on the surface of GCE. 31 The electric charge of DMAPS in solution is variable with the pH value. Fig. 1. shows the conductivity of DMAPS solutions with different pH values. It clearly shows that the isoelectric point (pI) of the polyelectrolyte DMAPS is about 4.1 from which it's easy to know that both DMAPS (PI 4.1) and GOX (pI 4.2) 32 are negatively charged at pH 6.98. Since the black dispersion of PDDA-CNTs is positively charged, the alternate adsorption of PDDA-CNTs, GOX and DMAPS-GR will result in alternative layers. The self-assemble process is schematically depicted in Scheme 2.
The bacteriostasis property of the sensor depends on the bacteriostasis compounds DMAPS. The bacteriostasis property of DMAPS can be shown by measuring the turbidity of the cell suspension, which is characterized by the optical density at a wavelength of 600 nm (OD 600 ). This is a common method for estimating the concentration of bacterial or other cells in solution. Fig. 2 shows the OD 600 curves of bacteria solution (E. Coli) with or without DMAPS. The higher OD 600 means the solution is opacity, and has more bacteria. Hence, lower OD 600 means better antibacterial activity. 33 If there is no DMAPS, the OD 600 will be high, and with DMAPS (10 wt%), the OD 600 will be lower. This demonstrates that DMAPS have strong bacteriostasis property against E. Coli.
Electrochemical characterization of the modified GCE electrode.-To study the electrochemical properties of the prepared biosensor, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are carried out in 5 mM Fe(CN) 6 3−/4− solution. Cyclic voltammetry in [Fe(CN) 6 ] 3−/4− solution is a convenient and valuable tool to determine the electrocatalytical activity of the surface modifier on the electrode. In this paper, cyclic voltammetry is carried out in 5 mM [Fe(CN) 6 ] 3−/4− solution at 50 mV s −1 . Fig. 3 shows the CV curves of the three electrodes: bare GCE, PDDA/GCE and PDDA-CNTs/GCE. The reduction peaks and oxidation peaks appear when the redox couples of [Fe(CN) 6 ] 3−/4− for the bare GCE is +0.27 V vs. SCE in forward scans and +0.04 V vs. SCE in reverse scans. As to the PDDA/GCE, the oxidation current decreases obviously compared with the bare GCE. While for the PDDA-CNTs/GCE, the oxidation peak current of [Fe(CN) 6 ] 3−/4− is larger than that at bare GCE. Furthermore, the reduction peak potential shifts more positive and the oxidation peak potential shifts more negative compared with the bare GCE. The larger peak current of CDP-CNT/GCE is caused by the larger surface area and electrocatalytic activity of CNTs dispersed in the modifying layer. EIS is a powerful method for the study of interface properties. 34 It can provide abundant information about the electrode surface as the impedance changes during the biosensor fabrication process. The charge-transfer resistance (Rct), which can indicate the electrontransfer kinetics of the redox probe (Fe(CN) 6 3−/4− ) on the electrode interface, could be estimated by the diameter of the semicircular part of EIS curve. 35  trode. The diameter of the semicircle of the EIS curve of (PDDA-CNTs/GOX) 4.5 /DMAPS-GR/GCE sensor is obviously larger than that of (PDDA-CNTs/GOX) 4 /GCE sensor, which provides direct experimental evidence about the successful deposition of DMAPS-GR on (PDDA-CNTs/GOX) 4 Fig. 5 shows the variation of response current of the prepared biosensors modified with different layers of PDDA-CNTs/GOX in 5.0 mM Glucose solution (pH 6.98) at + 0.6 V (vs.SCE). With the increase of PDDA-CNTs/GOX layers, when layers of PDDA-CNTs/GOX are less than four, the response current increases monotonously. However, when layers of PDDA-CNTs/GOX are more than four, the response current become decreasing monotonously. The four-layer PDDA-CNTs/GOX modified PDDA-CNTS/GOX/DMAPS-GR sensor has the highest performance.

Optimization of (PDDA-CNTs/GOX) n+0.5 /DMAPS-GR sensor.-
These kinds of biosensors are based on the oxidation of hydrogen peroxide generated according to the following reactions: β-D-glucose + O 2 + H 2 O glucose oxidase −→ D-gluconic acid + H 2 O 2 . Next, one of the products H 2 O 2 is decomposed on the electrode and produces response current. More layers have more enzymes and can produce more H 2 O 2 at the same time under the same condition. This may lead to higher response current. In the meantime, the mass transfer of glucose and H 2 O 2 is sensitive to distance. With the increase of layers, the mass transfer is becoming more and more difficult. This leads to rapid decrease of reaction rate and thus causes lower response current. 38 These two contrary effects result in the phenomenon that the four layers of biosensor have the highest response current.
Amperometric response to glucose.-The amperometric responses of the biosensor toward successive addition of glucose are measured by using a CHI660E electrochemical workstation. The working electrode is adjusted to be +0.60 V vs. SCE. The i-t curve is shown in Fig. 6. The biosensor can respond to the change of glucose concentration quickly and then reaches to a steady-state signal within 10 s. The resulting calibration plot for glucose over the concentration ranging from 0.0 mM to 253.6 mM is presented in the inset of Fig. 6. It shows that such an biosensor could work linearly from 16.5 to 214.3 mM. The corresponding regression equation of the linear plot is: i/μA = 2.50 + 0.07 c/mM, R = 0.99. The sensitivity is thus estimated as 70 nA mM −1 . The detection limit is estimated to be 3.5 mM (S/N = 3) according to the calibration curve. Table I shows the linear range (mM), limit of detection (μM), and applied potential (V vs. SCE) of some typical glucose sensors. The above parameters at this work are comparable with those mentioned  glucose sensors. Especially, the glucose sensor in present work shows very broad linear range. The interference measurement is carried out by adding possible interfering species, such as uric acid, ascorbic acid and dopamine with physiological concentration. These possible interferents do not substantially change the response signal of glucose. This may benefit from the relative lower sensitivity of the sensor.

Conclusions
In this paper, the multilayer films (PDDA-CNTS/GOX) n , are fabricated on the surface via electrostatic self-assembly. The GOX immobilized in the (PDDA-CNTS/GOX) 4 films displayed excellent electro catalytic activity to the reduction of Glucose. GR and DMAPS combined with (PDDA-CNTS/GOX) 4 are used to construct (PDDA-CNTS/GOX) 4.5 /DMAPS-GR sensor. The bacteriostatic properties of the sensor are ensured by the bacteriostatic characteristic of DMAPS. The (PDDA-CNTS/GOX) 4.5 /DMAPS-GR glucose sensor has a linear range from 16.5 to 214.3 mM. This study may provide a new stratagem for online monitor the bio-fermentation process.