Magnesium Zinc Oxide Nanostructure-Modified Multifunctional Sensors for Full-Scale Dynamic Monitoring of Pseudomonas aeruginosa Biofilm Formation

The MZO DGTFT biosensor has an MZO DGTFT as the signal transducer and an MZO_nano modified sensing pad as the biological receptor. The detailed fabrication process of the DGTFT biosensor was described elsewhere. ¹⁸ Here, we briefly summarize it as follows: first, a 50 nm Cr bottom gate layer was deposited and patterned on a 0.4 mm thick commercial glass substrate (PG&O), after which a 100 nm thick SiO₂ bottom gate dielectric was formed by plasma enhanced chemical vapor deposition (PECVD) at 250 °C. Next, a 5 nm ultra-thin MgO diffusion barrier ^8,10 followed by a 40 nm Mg_0.03Zn_0.97O active channel layer were deposited sequentially by metal oxide chemical vapor deposition (MOCVD) at ∼400 °C and fixed at width/length (W/L) = 160 μm/15 μm by wet etching. DeZn (Diethylzinc), MCp2Mg (Bis-(methylcyclopentadienyl)magnesium), and ultra-high purity (99.999%) oxygen gas were used as the Zn metalorganic source, Mg metalorganic source, and oxidizer, respectively. Then, the source/drain electrode (85 nm Ti/35 nm Au/5 nm Ti) was formed using the lift-off process. The SiO₂ top dielectric was subsequently deposited using PECVD with a thickness of 70 nm at the same temperature. A 150 nm thick Al top gate electrode was then formed by the electron beam evaporator and wet etching. VIA openings were finally made using diluted buffered oxide etch solution.

The fabrication process of the extended sensing pad started with the deposition of a 5 nm Cr/ 50 nm Au metal layer as the conducting electrode on the same kind of glass substrate. Then, the metal layer was etched to form squares of 120 mm² and followed by the growth of MZO_nano using MOCVD. Finally, the nanostructured layer was patterned and wet etched, while leaving a small area of the metal layer exposed for electrical contact.

The left side of Fig. 1b shows the cross-sectional schematic drawing of the MZO DGTFT and its testing configurations. During testing, the top gate of DGTFT was electrically connected to the exposed electrode of the extended sensing pad. The sensing pad was immersed in the bacterial incubator.

The MZO_nano QCM device consists of an MZO_nano modification layer that is integrated with a commercial AT-cut QCM (International Crystal Manufacturing, Inc.) by growing them directly on the surface of the sensing electrode of the QCM using MOCVD. The growth conditions of the MZO_nano are described below. A schematic drawing of the top and cross-sectional multilayer view of the MZO_nano QCM is shown on the right side of Fig. 1b. The standard QCM's quartz layer is sandwiched by two 100 nm gold electrodes. The sensing area of the QCM is 20.47 mm². The operation frequency of the standard QCM is 10 MHz, while the MZO_nano QCM has an operating frequency of 9.912 MHz.

The MZO_nano layers were deposited on the top electrodes of QCM and the extended pad of DGTFT to serve as the sensing surfaces to enhance their sensitivity. The 400 nm thick Mg_0.04Zn_0.96O nanostructured films were grown using MOCVD at ∼500 ℃ and the chamber pressure was maintained at ∼60 Torr. ²⁰ DeZn, MCp₂Mg, and high purity oxygen gas were used as the precursors and oxidizer, which are the same as the ones used for the DGTFT's MZO channel layer growth. Then the MZO_nano surfaces underwent UV illumination to achieve superhydrophilic characteristics to enhance the sensitivity and reduce the consumption of liquid biological samples. ²¹ The nanostructured surface provides a giant effective sensing area for bacteria to attach. In addition, as bacteria prefer to start adhesion at somewhere sheltered from shear forces, ²² the proper surface roughness also provides such optimum condition.

P. aeruginosa PAO1 was inoculated into Mueller Hinton Broth (MHB; Fisher Scientific) and grown at 37 °C for 16−18 h in test tubes placed in a shaking incubator operating at 200 rpm. The stationary phase cultures were diluted 100-fold into the fresh MHB medium and then pre-loaded in the Teflon cell culture well. The extended MZO_nano sensing pads of DGTFT and the MZO_nano QCMs were sterilized and placed into the wells. The biofilms were grown in the wells and the growing process was monitored by the DGTFT and the MZO_nano QCM.

We used the crystal violet staining to verify biofilm formation on the MZO_nano surfaces. MZO_nano was deposited on glass substrates and then divided into two sets: one for control and one for testing. Both sets were prepared through the same procedure as outlined above, however, for the control set the MZO_nano glass substrates were incubated with only MHB growth medium lacking bacteria. The separate Petri dishes containing the control and testing sets each containing multiple samples were placed in a static incubator at 37 °C to induce biofilm formation. At various time points during incubation, one sample each from the control and testing sets was retrieved for crystal violet staining. The time points of retrieval were 0, 1.7 h (100 min), 3.3 h (200 min), 5 h (300 min), 8 h, 15 h, and 24 h, which covers the biofilm development at different times. After removal from the culture, the samples were washed three times with 5 ml of 0.9% NaCl solution to remove planktonic cells to assure that only the biofilms were attached to the sample surface. Biofilms attached on the sensing surface were then stained with 0.2% crystal violet for 10 min, followed by washing three times with 0.9% NaCl. The microscopic images were then taken for each time point.

In the first step of measurement, the basic electrical characteristics of the MZO DGTFT were examined. The transistor's transconductance plots (i.e. drain current I_DS vs bottom gate biasing voltage V_BG) were recorded under various top gate biasing voltages V_TG. Then, the control experiment with MHB medium only was tested three times for 660 min. The results showed an average drain current variation of 1.6% with an average standard deviation of 2.9%. Comparing with the actual biosensing results which will be discussed later, such background variations are negligible. Next, the transfer characteristic variations, as a result of the early stage detection of P. aeruginosa biofilm formation, were obtained. As the electrons from bacterial membrane transfer to the MZO_nano, an equivalent micro biasing voltage is applied to the top gate of the DGTFT. Such top gate bias changes the conducting current I_DS flowing through the channel layer through the electric field effect. By extracting the current variations under proper bottom gate biasing V_BG, the optimized signal output can be realized with the best combination of high sensitivity and stability. At last, the time-dependent signal response with standard deviation error bars is presented in combination with the QCM's results to form the full-scale monitoring results.

Throughout the experiments, the MZO DGTFT was placed inside a light-tight measurement station. Its top gate was electrically connected to the extended MZO_nano sensing pad where the bacterial incubation took place. The electrical measurements were carried out using the HP-4156C semiconductor parameter analyzer. The V_BG was swept between −5 V to 15 V to find the optimum operation condition. The voltage across source and drain V_DS acts as an electron pump to drive current I_DS across the channel and was maintained at 0.1 V to minimize power consumption. The device threshold voltage V_TH was extracted using the linear fitting method for 10%−90% of the maximum drain current. The subthreshold slope S.S value was extracted from a 3-decade range in the sub-threshold region (I_DS = 10⁻¹² − 10⁻⁹ A) of the transconductance curve in logarithm scale.

The MZO_nano QCM was used to monitor the long-term (24 h) development process of P. aeruginosa biofilms. The device was placed in a standard bacterial incubator with a controlled ambient environment. The characterization of MZO_nano QCM was conducted using an HP-8573D network analyzer, which was connected via the IEEE-488 general purpose interface bus (GPIB) to the universal serial bus (USB) of a microprocessor running of a LabView data acquisition program. The impedance transmission spectrum Z₂₁ (ω) of the device was automatically measured at fixed time intervals and digitally stored. The MZO_nano QCM was sterilized and deployed inside a Teflon cell-growth well seeded with growth media. Both frequency shift and motional resistance keep slightly increasing until equilibrium at 40 min. Then, P. aeruginosa cells were added to the device. Background signal variations were excluded during the biosensing process.

The impedance spectrum of the MZO_nano QCM can provide the biophysical properties of the bacterial culture as a function of time. The spectrum features two important parameters, namely the peak frequency shift (Δf) and the motional resistance (R_load). The frequency shift parameter Δf of the MZO_nano QCM is determined by measuring the absolute value of the difference in resonant frequency between the case without and with mass loading. ²³

$$\begin{eqnarray}&&{\rm{\Delta }}f=\displaystyle \frac{2{{f}_{0}}^{2}}{{\nu }_{q}{\rho }_{q}A}{\rm{\Delta }}m\end{eqnarray} \tag{ 1 }$$

where for Eq. 1, Δf = f₀ − f(t) is the frequency shift and f₀ is the intrinsic operating frequency of the device, υ_q and ρ_q are respectively the acoustic velocity and mass density of the AT-cut quartz layer, A is the sensing area of the top electrode, and Δm is the mass change, i.e. the accumulated mass on the sensing electrode.

The motional resistance R_load is obtained from the real part of the complex amplitude in the impedance transmission spectrum, i.e. R_load = Re{Z₂₁(ω)}. With the development of biofilm on the MZO_nano QCM sensing surface, the biofilm undergoes viscoelastic transitions due to the attachment and metabolism activities of bacterial cells and the formation of EPS. These viscoelastic transitions manifest themselves as modulations in the amplitude of the impedance transmission spectrum, specifically on R_load. These two parameters can be represented by the equations below, respectively. ^13,24

$$\begin{eqnarray}&&{R}_{Load}=\displaystyle \frac{\pi }{4{K}^{2}{\omega }_{0}{C}_{0}{Z}_{BAW}}{\rm{Re}}\left\{{Z}_{mechL}\right\}\end{eqnarray} \tag{ 2 }$$

where K² is the coupling coefficient of the piezoelectric layer, ω₀ is the resonant angular frequency of the QCM with no mass loading, C₀ is the capacitance of the device, Z_BAW is the impedance of the device without the mass accumulation, and Z_mechL is the mechanical load impedance due to the biofilm.

The transfer characteristics of the MZO DGTFT were firstly tested with its top gate electrode electrically connected to a DC power supply. We chose V_TG from 0 to −1 V with a step of −0.2 V as the setting to demonstrate the highly sensitive signal of the device in response to the voltage alternation on its top gate electrode. Such V_TG was chosen because the bacteria tend to donate only a small fraction of their membrane electrons to the supporting substratum, ^25,26 and thus the equivalent top gate bias induced by bacterial adhesion should be negative and small.

The measured results are shown in Fig. 2a. The curve at V_TG = 0 V shows the threshold voltage of 7.47 V, and subthreshold slope of 637 mV dec⁻¹. The low threshold voltage ensures the device with low power consumption. The steep subthreshold slope enables high sensitivity owing to the high electrical signal gain of the device. To have a closer look at the variations of the I-V curves due to different top gate biases, the inset figure in Fig. 2a shows the detailed characteristics in the range of -3 V < V_BG < 2 V where the device is just about to be turned on. It could be clearly seen that these transfer curves exhibited parallel right shifts with respect to the increasing values of V_TG. The threshold voltage V_TH positively shifts ∼10% from 7.47 V at V_TG = 0 V to 8.25 V at V_TG = -1 V. Such right shifting can be explained as follows:

MZO is a kind of n-type semiconductor material. The bottom gate bias introduces a vertical electrical field that accumulates electrons at the bottom channel/dielectric interface. However, the negative top gate voltage partially depletes the accumulation channel. To compensate the depletion and turn on the device, the threshold voltage must be adjusted by an equivalent positive shift as shown in Fig 2.

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An equation (Eq. 3) describing the relationship between V_TH and V_TG was proposed by G. Baek et al. ²⁷

$$\begin{eqnarray}&&{V}_{TH}={V}_{TH}\left(0\right)+\beta {V}_{TG}\end{eqnarray} \tag{ 3 }$$

where V_TH(0) denotes the threshold voltage when V_TG = 0, and β is the coefficient related to the capacitance of dielectric layers and the channel layer. Based on the measurement result, the β value is calculated to be 0.78.

The right shifting of threshold voltage also lowers the drain current under a certain bottom gate biasing condition, especially in the triode region of the transfer characteristics. For example, the drain current at V_BG = 0 V decreases nearly one decade from 1.37 × 10⁻⁹ A at V_TG = 0 V to 1.41 × 10⁻¹⁰ A at V_TG = −1 V (shown in the inset of Fig. 2a). The steep slope in the triode region of the transfer characteristics results in large current variations within a small range of bottom gate biases, and hence provides high current amplification when the top gate bias varies. Comparing to the threshold voltage change, the drain current change of DGTFT is particularly favorable for using as the signal to represent the early stage development of biofilm formation and is used to define the sensitivity of the biosensor.

The high electrical sensitivity of the device enables the DGTFT to detect the early stage of biofilm formation. Next, the MZO DGTFT biosensor was used to detect the onset of P. aeruginosa biofilm formation with its top gate electrode electrically connected to the sensing pad where bacterial growth occurs. Three MZO_nano modified sensing pads were prepared. The detection of P. aeruginosa biofilms was performed on each of these pads using the same DGTFT device under the same microbial culture and measurement conditions. The sensing pad was immersed in the MHB medium and was allowed its baseline signal to stabilize before the P. aeruginosa culture solution was introduced at time t = 0. The measurements of transfer characteristics were made sequentially for a total of 900 min of incubation time.

In obtaining the signal that represents the status of early stage biofilm development, we've described how to balance between sensitivity and stable operations when choosing the best point of operation. ¹⁸ Using a similar method, V_BG = 5 V is determined as the optimum operation point in this study. For better visualization, part of the transfer characteristics (2.5 V < V_BG < 7.5 V) of a single set of measurements are shown in Fig. 2b. As can be clearly seen from the figure, the drain current keeps dropping until t = 390 min where it exhibits no significant variation comparing with the one at t = 900 min DGTFT is a highly sensitive electrical device. Drain current drops as a result of the negative bias applied on its top gate electrode. In the biosensing experiment, the current change is attributed to the electron charge transfer resulted from the bacterial adhesion to the MZO_nano surface during the early stage development of the biofilm formation. ^25,26

The testing results show no significant signal variation after t = 390 min, indicating limited long-term monitoring capability of the device. Thus, the biofilm development process beyond the early stage is essentially difficult to detect using this MZO DGTFT biosensor. This prompted us to employ the MZO_nano QCM for monitoring the long-term biological evolutions of biofilm formation to complement MZO DGTFT for monitoring the later stages of biofilm formation.