Development of an Enzyme-Coated Microcantilever-Based Biosensor for Speciﬁc Detection of Short-Chain Alcohols †

: This paper describes the development of a biosensor designed for the enzymatic detection of short-chain alcohols. The biorecognition element, alcohol dehydrogenase, was immobilized on self-assembled monolayers deposited on top of silicon nitride microcantilevers. The self-assembly process was performed by surface activation using 3-aminopropyltriethoxysilane, followed by glutaraldehyde and biomolecule binding. X-ray photoelectron spectroscopy and atomic force microscopy were used. The biosensor showed a lower response time and sensibility from 0.03 to 1.2 mL/L. Its selectivity was analyzed through exposure to pure and mixed volatile solvents. Sensor sensibility was higher in the presence of short-chain alcohols and practically null involving other polar or nonpolar solvents.


Introduction
Alcohols are important compounds in medicine, biotechnology and mainly in the food industry, in which some procedures may involve fermentation and distillation. However, in some cases, the volatilized concentration of alcohols can reach toxic levels, causing inflammation of the nasal and conjunctiva mucous membrane, skin irritation and poisoning, besides being highly flammable. Given these circumstances, monitoring the volatilized alcohol concentration in the air is important [1,2]. In nature, the detection of methanol can signal plant immunity, with potential application in plant phenotyping [3].
Detection of small quantities of VOC in a gaseous medium requires a sensitive sensor. In this context, the development of microcantilever (µC)-based biosensors has been an efficient solution [4,5]. Microcantilevers are mechanical probes with a special format used to measure small forces, and different probes are employed for investigations with atomic force microscopes (AFM) [6].
The initial application of a microcantilever as a sensor involved a mass-sensitive balance, which acted as a microresonator, reaching resolutions in the order of picograms and allowing the detection of individual virus particles [7]. Microcantilevers with a high Q factor-in the order of 10,000-and with high-frequency operation-around 1.5 MHz-allow a resolution of theoretical mass of around 20 ag/Hz [8]. Usually, sensors translate the change in a physical property into measurable electrical signals; however, in this study, the mechanical response of a µC was examined through AFM. The immobilization of biomolecules, such as enzymes, in a sensor promotes the affinity and high selectivity of catalytically active proteins for the 2 of 5 detection of a specific target, and studies applying alcohol dehydrogenase enzyme immobilization have been reported for the detection of alcohols using amperometric [1,9,10] and voltammetric sensors [11,12]. The enzymatic biosensors offer a combination of performance and analytical features not available in other bioanalytical systems [13]. Microcantilevers can be coated on both sides or just one side with a biosensitive layer in a process called functionalization, making it possible to detect the mass variation in the set by changes in the resonant frequency. This variation of the technique is commonly used in liquid media [14].

Immobilization of Biomolecules on Microcantilever Surfaces
Immobilization of biomolecules on microcantilever surfaces can be seen as closely related to the immobilization methods used to fabricate electrodes; these procedures are gathered under the generic term "chemically modified devices" [15].
In this paper, we present a specific enzymatic biosensor that uses a signal transduction based on mechanical displacement, which differs from commonly commercially available voltammetric and resistive sensors.

Reagents
All chemicals and buffer components were used as received. The solvent was provided by J.T. Baker and other products such as APTES, triethylamine and glutaraldehyde (GLD) were used as received from Sigma Aldrich, St. Louis, MO, USA.

Microcantilevers (µCs)
Silicon nitride microcantilevers used were HA_NC model (NT-MDT) with stems at both ends, with (A) being the shortest and (B) the longest, as Table 1 shows. Microcantilevers were subjected to heat treatment at 500 • C for eight hours, subsequently washed with piranha solution and then extensively washed with milli-Q water to remove excess solution. After this process, the microcantilevers were subjected to an activation procedure through vaporization of 40 µL of APTES and 40 µL of triethylamine in a nitrogen atmosphere for one hour [16].

Biomolecule Binding
The functionalization through the formation of the self-assembled monolayer (SAM) activated with APTES [16,18,19] was performed with alcohol dehydrogenase enzyme stock solution (0.25 mg/mL) dissolved in 50 mM sodium phosphate buffer, pH 8.6.

Instrumentation (Scanning Probe Microscopy (AFM))
The frequency response of the microcantilevers during excitation was measured using a Veeco Dimension V AFM.

X-ray Photoelectron Spectroscopy (XPS)
The XPS spectra were acquired for identifying and quantifying all the chemical elements on the surface of the sample (µC), using a spectrometer from Scienta Omicron.

Results and Discussion
In Figure 1a, we present a comparison of the response of a bare (Control) and functionalized µC when exposed to vapor of 10 µL of ethanol; the figure shows the short response time of the biosensor of less than 1 s (A); in (B), it shows the influence of the surface tension and depicts the adsorption as well as the total recovery of the bioactive layer after 10 min (C). Figure 1c shows the sensibility ranging from 0.03 to 1.2 mL/L. To verify the reproducibility of the measurement (Figure 1b), it was carried out three times, using the same experimental conditions given in Figure 1a.

Biomolecule Binding
The functionalization through the formation of the self-assembled monolayer (SAM) activated with APTES [16,18,19] was performed with alcohol dehydrogenase enzyme stock solution (0.25 mg/mL) dissolved in 50 mM sodium phosphate buffer, pH 8.6.

Instrumentation (Scanning Probe Microscopy (AFM))
The frequency response of the microcantilevers during excitation was measured using a Veeco Dimension V AFM.

X-ray Photoelectron Spectroscopy (XPS)
The XPS spectra were acquired for identifying and quantifying all the chemical elements on the surface of the sample (µC), using a spectrometer from Scienta Omicron.

Results and Discussion
In Figure 1a, we present a comparison of the response of a bare (Control) and functionalized µC when exposed to vapor of 10 µL of ethanol; the figure shows the short response time of the biosensor of less than 1 s (A); in (B), it shows the influence of the surface tension and depicts the adsorption as well as the total recovery of the bioactive layer after 10 min (C). Figure 1c shows the sensibility ranging from 0.03 to 1.2 mL/L. To verify the reproducibility of the measurement (Figure 1b), it was carried out three times, using the same experimental conditions given in Figure 1a.  The selectivity was analyzed through exposure to pure and mixed volatile solvents ( Figure 2). The sensor's sensibility was higher in the presence of short-chain alcohols (methanol, ethanol and propanol), ranging from 0.45 to 0.85 kHz, and practically null involving other polar or nonpolar solvents.
The selectivity was analyzed through exposure to pure and mixed volatile solvents ( Figure 2). The sensor's sensibility was higher in the presence of short-chain alcohols (methanol, ethanol and propanol), ranging from 0.45 to 0.85 kHz, and practically null involving other polar or nonpolar solvents.

Conclusions
The functionalization of microcantilevers with alcohol dehydrogenase enzyme immobilized in self-assembled monolayers allowed the construction of a biosensor for the selective detection of short-chain alcohols, at ambient conditions, even in the presence of a mixture of VOCs. The biosensor was evaluated using AFM, with dynamic mode and contact mode, to obtain 3D images of the surface, as well as XPS to identify and quantify all the chemical elements on the surface of the µC. The biosensor developed showed less susceptibility to humidity and temperature variations, presenting high quality, a faster response time, and high selectivity, sensitivity and durability.

Conclusions
The functionalization of microcantilevers with alcohol dehydrogenase enzyme immobilized in self-assembled monolayers allowed the construction of a biosensor for the selective detection of short-chain alcohols, at ambient conditions, even in the presence of a mixture of VOCs. The biosensor was evaluated using AFM, with dynamic mode and contact mode, to obtain 3D images of the surface, as well as XPS to identify and quantify all the chemical elements on the surface of the µC. The biosensor developed showed less susceptibility to humidity and temperature variations, presenting high quality, a faster response time, and high selectivity, sensitivity and durability.