Staphylococcus aureus Detection in Milk Using a Thickness Shear Mode Acoustic Aptasensor with an Antifouling Probe Linker

Contamination of food by pathogens can pose a serious risk to health. Therefore, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination of food. In this work, an aptasensor based on a thickness shear mode acoustic method (TSM) with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow’s milk. The frequency variation and dissipation data demonstrated the correct immobilization of the components. The analysis of viscoelastic properties suggests that DNA aptamers bind to the surface in a non-dense manner, which favors the binding with bacteria. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the sensor’s antifouling properties, which is based on 3-dithiothreitol propanoic acid (DTTCOOH) antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), and 1-undecanethiol (UDT)) quartz crystals, the sensitivity of the sensor’s antifouling in milk improved by about 82–96%. The excellent sensitivity and ability to detect and quantify S. aureus in whole UHT cow’s milk demonstrates that the system is applicable for rapid and efficient analysis of milk safety.


Introduction
Nutrition is a fundamental process in human life as it allows the body to obtain nutrients necessary for growth and survival. However, food can pose a risk to human health such as through its potential contamination with pathogens [1]. Milk is no exception; each step of its processing, from milking to distribution, can be affected by microbiological contaminants. Monitoring food for the presence of pathogens is important for identifying and regulating such contamination [2].
There are numerous bacteria that can be found in milk depending on different handling processes of food [2]. Immediately after milking, microorganisms in the milk come largely from environmental contamination and their concentrations can change during processing and manipulation [2]. There are many dangerous pathogenic bacteria capable of growing in milk and dairy products: in particular, those of the genus Campylobacter, Listeria, Salmonella, Brucella, Mycobacterium, Staphylococcus, Clostridium, Bacillus, and Pseudomonas.
Staphylococcus aureus (S. aureus) bacteria and their toxins can cause serious infections such as sepsis. S. aureus bacteria can be found in the environment, such as in dust or soil, and on living organisms. As the bacteria is present in normal human flora, individuals can also be a contamination source for food [3]. The low acidity and high protein content of milk provides an ideal environment for the rapid growth of S. aureus. Although cooking milk to in this work, we applied TSM with dissipation monitoring that provides information about dissipation, which is the energy that is lost due to variation in a viscoelastic layer. Dissipation is proportional to the damping of the resonance. As damping increases, more energy is dissipated, which indicates that a more viscoelastic layer has been adsorbed on the surface. For preparation of the aptasensor sensitive to Staphylococcus aureus, we used the same linker as in the previous work, namely, DTT COOH [10]. We also analyzed DTT COOH by Fourier transform infrared spectroscopy (FTIR) for functional group and structure confirmation. However, to improve the antifouling properties of the sensing layer, the aptasensor's SAM layer also included 2-(2-mercaptoethoxy)ethan-1-ol (HS-MEG-OH), a previously synthesized antifouling molecule [13]. HS-MEG-OH was used instead of β-mercaptoethanol as its ether moiety allows for the incorporation of interfacial water molecules. In addition, we also analyzed the viscoelastic properties of the aptamer-based sensing layers.

Materials
The synthesis of 3-dithiothreitol propanoic acid (DTT COOH ) and HS-MEG-OH ( Figure 1) followed previously published methods [12,14]. The functional groups of DTT COOH were analyzed by FTIR (see Supplementary Material, Figures S1 and S2). Sodium chloride, absolute analytical grade ethanol, hydrogen peroxide (30% in water w/w), 1-undecanethiol (UDT), 11-mercaptoundecanoic acid (MUA), dithiothreitol (DTT), N-hydroxysuccinimide (NHS), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), and ethanolamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water (specific resistance of 18.2 MΩ·cm) was used for preparing aqueous solutions. Ethanol was purchased from Caledon Laboratory Chemicals (Georgetown, ON, Canada). The chemicals were used without purifying. of the viscoelastic contribution in bacteria-aptamer interactions. Therefore, in this work, we applied TSM with dissipation monitoring that provides information about dissipation, which is the energy that is lost due to variation in a viscoelastic layer. Dissipation is proportional to the damping of the resonance. As damping increases, more energy is dissipated, which indicates that a more viscoelastic layer has been adsorbed on the surface. For preparation of the aptasensor sensitive to Staphylococcus aureus, we used the same linker as in the previous work, namely, DTTCOOH [10]. We also analyzed DTTCOOH by Fourier transform infrared spectroscopy (FTIR) for functional group and structure confirmation. However, to improve the antifouling properties of the sensing layer, the aptasensor's SAM layer also included 2-(2-mercaptoethoxy)ethan-1-ol (HS-MEG-OH), a previously synthesized antifouling molecule [13]. HS-MEG-OH was used instead of β-mercaptoethanol as its ether moiety allows for the incorporation of interfacial water molecules. In addition, we also analyzed the viscoelastic properties of the aptamer-based sensing layers.
The preparation of phosphate-buffered saline (PBS) involved 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 at pH 7.4. The buffer was filtered with a

Cleaning and Surface Modification of Piezocrystals
The AT-cut quartz crystals (0.2 cm 2 sensing area, 8 MHz fundamental frequency) were purchased from Total Frequency Control Ltd., Storrington, UK. The crystals had gold electrodes deposited on both sides. Basic Piranha solution (7 mL of 1:1:5 v/v 28-30% NH 4 OH, 30% H 2 O 2 , Milli-Q water at 70 • C) was used to clean each crystal in three 25 min cycles. In between the cycles, the crystals were rinsed with Milli-Q water three times. After the third Piranha cycle, the crystals were rinsed with Milli-Q water twice, then with methanol twice.
After cleaning, the quartz crystals were functionalized in a solution of 2 mM UDT, 2 mM MUA, 50 µM DTT, or 50 µM DTT COOH in absolute ethanol overnight. For DTT COOHcoated crystals, the surfaces were further modified in 2 mM HS-MEG-OH in absolute ethanol (25 min The preparation of phosphate-buffered saline (PBS) involved 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 at pH 7.4. The buffer was filtered with a 0.22 µm membrane (Merck-Millipore, Darmstadt, Germany). Staphylococcus aureus KR3 was purchased from the University of Toronto Medstore (Toronto, ON, Canada). Specificity experiments were carried out with Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. Whole UHT cow milk (3.5% fat) was bought from Walmart (Toronto, ON, Canada).

Cleaning and Surface Modification of Piezocrystals
The AT-cut quartz crystals (0.2 cm 2 sensing area, 8 MHz fundamental frequency) were purchased from Total Frequency Control Ltd., Storrington, UK. The crystals had gold electrodes deposited on both sides. Basic Piranha solution (7 mL of 1:1:5 v/v 28-30% NH4OH, 30% H2O2, Milli-Q water at 70 °C) was used to clean each crystal in three 25 min cycles. In between the cycles, the crystals were rinsed with Milli-Q water three times. After the third Piranha cycle, the crystals were rinsed with Milli-Q water twice, then with methanol twice. After

Contact Angle Goniometry
Static contact angle goniometry (CAG) experiments were conducted with the KSV CAM 101 goniometer (KSV Instruments Ltd., Helsinki, Finland). A 5 µL drop of Milli-Q water at room temperature was used for each measurement. Bare and coated TSM crystals were analyzed in triplicate.

Bacteria Preparation
Lysogeny broth was used to grow S. aureus bacteria at 37 °C overnight. The grown solution was serially diluted from 1/10 to 1/10 9 times in PBS. Each solution was spotted onto agar plates (3 × 10 µL), as well as measured using a UV-1600PC spectrometer (VWR International, Mississauga, ON, Canada) to measure the optical density at 600 nm (OD600). The plates were incubated overnight at 37 °C and spot counted to calculate CFU per OD600 (see Supplementary Material, Section S3).

Contact Angle Goniometry
Static contact angle goniometry (CAG) experiments were conducted with the KSV CAM 101 goniometer (KSV Instruments Ltd., Helsinki, Finland). A 5 µL drop of Milli-Q water at room temperature was used for each measurement. Bare and coated TSM crystals were analyzed in triplicate.

Bacteria Preparation
Lysogeny broth was used to grow S. aureus bacteria at 37 • C overnight. The grown solution was serially diluted from 1/10 to 1/10 9 times in PBS. Each solution was spotted onto agar plates (3 × 10 µL), as well as measured using a UV-1600PC spectrometer (VWR International, Mississauga, ON, Canada) to measure the optical density at 600 nm (OD600). The plates were incubated overnight at 37 • C and spot counted to calculate CFU per OD600 (see Supplementary Material, Section S3).
For TSM measurements, S. aureus, E. coli, or P. aeruginosa bacteria was grown overnight at 37 • C. The next day, 1 mL of the bacteria solution was centrifuged at 14,500× g RPM (5 min). A total of 1 mL of PBS was used to resuspend the bacteria pellet. The OD600 of the solution was used to calculate the base CFU, and then the solution was diluted to the desired CFU. To prepare milk samples, the diluted PBS bacteria solution was centrifuged at 14,500× g RPM (5 min) to pellet the bacteria, and then the bacteria was resuspended in milk.

TSM Measurements
Cleaned or modified crystals were inserted in an acryl flow-through cell (JKU, Linz, Austria), which was clamped by a holder, ensuring the internal conductors were in contact with the crystal's electrodes. Liquid flowed through the internal chamber using a GeniePlus syringe pump (Kent Scientific, Torrington, CT, USA) and a pulling syringe. The liquid flowing through the internal chamber was in contact with one face of the crystal. A SARK-110 vector analyzer (Seeed, Shenzhen, China) was used to collect data via Python software [16]. Experiments were measured at 8 MHz under ambient conditions and under a 50 µL·min −1 constant flow. The scheme of experimental setup is presented in Supplementary Material ( Figure S5).
Marketed whole milk was used as a fouling agent for antifouling experiments. The crystals were first rinsed with PBS buffer to wash off weakly adsorbed thiol molecules and to reach a stable baseline (about 50 min). After baseline was achieved, the solution was changed to milk samples for bare, UDT, MUA, DTT, and DTT COOH crystals (250 µL over 5 min of flow) then returned to flow under PBS buffer.
For the aptasensor, DTT COOH -modified crystals were functionalized according to the same procedure described above (Section 2.2). The crystals were exposed to HS-MEG-OH (25 min), rinsed with PBS (5 min), activated with NHS/EDC (35 min), rinsed with PBS (5 min), incubated with aptamer solution (90 min), rinsed with PBS (5 min), incubated with ethanolamine solution (40 min), then washed with PBS (at least 15 min). HS-MEG-OH was used to functionalize any possible exposed gold on the crystal surface, while ethanolamine neutralized activated carboxyl groups that did not react with aptamer. Once the final PBS wash reached a stable baseline following the aptasensor functionalization, whole milk was flowed (250 µL over 5 min of flow) to analyze the crystal's antifouling character. Alternatively, for S. aureus detection, 250 µL of milk sample containing a known concentration of bacteria (10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 CFU/mL) was poured over the crystal surface. After milk (with or without bacteria), PBS buffer was poured over the crystals to wash them, remove any remaining sample on the surface, and reach a final stable baseline. Each experiment was repeated three times.

TSM Data Analysis
By detecting variations in frequency and dissipation, it is possible to obtain information on the mass deposited on the electrode of the crystal using the Sauerbrey equation [17]: ∆f (Hz) is the frequency change, n is the number of harmonics, and f 0 is the fundamental frequency. ∆m is the mass adsorbed on the surface, A is the active area of the quartz crystal electrode, µ q is the shear modulus, 2.947 × 10 10 Pa, and ρ q is the density, 2.648 × 10 3 kg m −3 , of quartz.
The sensor sensitivity has been evaluated by determination of the limit of detection (LOD) according to [18] as follows: where SD lc is the standard deviation at low concentration of the sample, and the limit of blank (LOB) is the highest apparent analyte concentration: Biosensors 2023, 13, 614 6 of 14 The limit of quantification (LOQ) has been calculated according to the equation: It is also possible to analyze changes in the viscoelastic properties following the functionalization of the thiol SAM with the aptamer in order to study the density of the bioreceptor anchored on the surface and confirm the success of the functionalization. In particular, the shear modulus, µ A , the viscosity coefficient, η A , and the penetration depth, Γ A , of the evanescent acoustic after the aptamer incubation wave were calculated. To perform this analysis, the Voinova-Voigt viscoelastic model was employed [19]: where χ = µ 1 η 1 ω ; Γ is the penetration depth of the shear wave in the liquid medium; ρ 0 is the density of quartz; h 0 is the thickness of the quartz crystal; h 1 , µ 1 , η 1 , and ρ 1 are the thickness, the shear elastic modulus, the viscosity, and the density of the adsorbed film, respectively; and ω = 2πf is the angular frequency of oscillation. DNA has an average density of 1.7 g × cm −3 , and the thickness can be obtained by dividing the calculated mass per unit area by the density.
A Python code based on Yoon et al.'s equation [20] was used to analyze the data. Excel Office 365 (Microsoft Corporation, Albany, NY, USA) and OriginPro 8 (OriginLab Corporation, Northampton, MA, USA) were then used to plot and statistically process the data. The aptasensor was incubated with various concentrations of S. aureus in triplicate.

Contact Angle Goniometry
Different SAM functionalizations of the gold electrode surfaces were confirmed with CAG. The same values were obtained as the previous work: approximately 55 • , 105 • , 48 • , 43 • , and 35 • for bare gold, and UDT-, MUA-, DTT-, and DTT COOH -modified crystals, respectively [12]. As MUA is hydrophilic, its wettability is higher compared to UDT and bare crystals [12]. UDT crystals showed low wettability compared to bare crystals as UDT is non-polar. The contact angle of DTT was similar to our previous work, as well as to that in the literature [12,21], which confirmed that the crystal was successfully functionalized as DTT-modified TSM discs are more polar than bare gold. As DTT COOH is a derivative of DTT, it was expected that DTT COOH -modified surfaces would also be hydrophilic, which was confirmed with CAG. As DTT COOH has a carboxylic acid group, this made DTT COOH SAMs more wettable compared to DTT SAMs.
As DTT SAMs have disordered binding to gold, DTT COOH likely experiences similar binding [22]. Such less dense SAMs can form vacancy islands due to rearranging gold atoms [23]. As a result, we used SH-MEG-OH as an antifouling linear thiol to functionalize any exposed areas of the gold surface.

Milk Antifouling Test with TSM Method
To determine the aptasensor's antifouling ability, different SAM-modified quartz crystals were exposed to marketed whole milk ( Figure 3). As whole milk contains high protein and fat content, it strongly adsorbs to nonpolar surfaces such as MUA and UDT SAMs. MUA, which is often used as a thiol linker on gold surfaces, experienced slightly less fouling compared to the more hydrophobic UDT SAM crystals.
SAMs. MUA, which is often used as a thiol linker on gold surfaces, experienced slightly less fouling compared to the more hydrophobic UDT SAM crystals. As Table 1 summarizes, UDT had the most fouling (158 ± 16 Hz and (8.0 ± 2.4) × 10 −6 frequency and dissipation shifts, respectively) as its structure is only hydrophobic, while MUA's hydrocarbon chain and carboxylic acid functional group caused slightly less fouling (136 ± 4 Hz and (7.6 ± 0.8) × 10 −6 frequency and dissipation shifts, respectively). Bare gold experienced high milk adsorption (105 ± 5 Hz and (6.1 ± 0.6) × 10 −6 frequency and dissipation shifts, respectively), which is less than for UDT-and MUA-functionalized discs. UDT, MUA, and bare gold demonstrated 64%, 58%, and 45% more fouling relative to DTTCOOH, respectively. Table 1. Frequency, Δf, and dissipation, ΔD, changes after exposure to whole milk and washing with running buffer. % fouling relative to DTTCOOH has been calculated as (Δf/ΔfDTTCOOH)x100, where Δf denotes the frequency changes for corresponding SAM surface. Average and standard deviations (SD) were determined from three independent experiments.

SAM
Δf ( In comparison, DTT and the aptasensor were, respectively, 31% and 88% less fouling than DTTCOOH-modified TSM crystals. DTT is likely less fouling (40 ± 8 Hz and (2.8 ± 1.4) × 10 −6 frequency and dissipation shifts, respectively) than DTTCOOH as it does not have a carboxylic acid group that can become negatively charged; instead the DTT SAM remains neutral in milk.
The aptasensor was significantly less fouling compared to DTTCOOH as the carboxylic acids are likely deprotonated when exposed to the slightly acidic milk environment. The negatively charged DTTCOOH SAM can electrostatically interact with positively charged species, which causes adsorption of milk (58 ± 14 Hz and (3.4 ± 2.7) × 10 −6 frequency and dissipation shifts, respectively). As the aptasensor's carboxylic acid groups are modified with aptamers or ethanolamine, the lack of exposed carboxylic acid groups significantly decreases fouling to a minimal amount (7 ± 1 Hz and (0.9 ± 0.2) × 10 −6 frequency and dissipation shifts, respectively). Modifying the DTTCOOH SAM to an aptasensor significantly As Table 1 summarizes, UDT had the most fouling (158 ± 16 Hz and (8.0 ± 2.4) × 10 −6 frequency and dissipation shifts, respectively) as its structure is only hydrophobic, while MUA's hydrocarbon chain and carboxylic acid functional group caused slightly less fouling (136 ± 4 Hz and (7.6 ± 0.8) × 10 −6 frequency and dissipation shifts, respectively). Bare gold experienced high milk adsorption (105 ± 5 Hz and (6.1 ± 0.6) × 10 −6 frequency and dissipation shifts, respectively), which is less than for UDT-and MUA-functionalized discs. UDT, MUA, and bare gold demonstrated 64%, 58%, and 45% more fouling relative to DTT COOH , respectively. In comparison, DTT and the aptasensor were, respectively, 31% and 88% less fouling than DTT COOH -modified TSM crystals. DTT is likely less fouling (40 ± 8 Hz and (2.8 ± 1.4) × 10 −6 frequency and dissipation shifts, respectively) than DTT COOH as it does not have a carboxylic acid group that can become negatively charged; instead the DTT SAM remains neutral in milk.
The aptasensor was significantly less fouling compared to DTT COOH as the carboxylic acids are likely deprotonated when exposed to the slightly acidic milk environment. The negatively charged DTT COOH SAM can electrostatically interact with positively charged species, which causes adsorption of milk (58 ± 14 Hz and (3.4 ± 2.7) × 10 −6 frequency and dissipation shifts, respectively). As the aptasensor's carboxylic acid groups are modified with aptamers or ethanolamine, the lack of exposed carboxylic acid groups significantly decreases fouling to a minimal amount (7 ± 1 Hz and (0.9 ± 0.2) × 10 −6 frequency and dissipation shifts, respectively). Modifying the DTT COOH SAM to an aptasensor significantly improves the layer's antifouling properties, as the neutral and polar surface is likely more effective for interacting with water molecules. Furthermore, the dithiol structure of DTT COOH provides spacing for the water molecules to hydrogen bond with the layer, creating a thermodynamically favorable "water barrier" and largely preventing milk components from adsorbing. Compared to DTT, bare gold, MUA, and UDT crystals, the aptasensor's fouling significantly decreased by 82%, 94%, 95%, and 96%, respectively.

Sensing of Staphylococcus Aureus in Milk
The aptasensor showed high sensitivity to S. aureus in milk. Different bacteria concentrations caused proportional decreases in the resonant frequency. The dissipation changes confirmed the sensitivity as they increased with increasing bacteria concentrations, indicating that S. aureus adsorbed to the crystal's surface by binding with the aptamer. As Figure 4 shows, the crystal experiences minimal frequency and dissipation shifts when exposed to milk without bacteria (8.9 ± 3.4 Hz and (2.1 ± 0.8) × 10 −6 frequency and dissipation shifts, respectively). Bacteria concentrations in milk were proportional to changes in the frequency and dissipation; increasing cell concentration caused decreasing frequency and increasing dissipation shifts.
improves the layer's antifouling properties, as the neutral and polar surface is likely more effective for interacting with water molecules. Furthermore, the dithiol structure of DTTCOOH provides spacing for the water molecules to hydrogen bond with the layer, creating a thermodynamically favorable "water barrier" and largely preventing milk components from adsorbing. Compared to DTT, bare gold, MUA, and UDT crystals, the aptasensor's fouling significantly decreased by 82%, 94%, 95%, and 96%, respectively.

Sensing of Staphylococcus Aureus in Milk
The aptasensor showed high sensitivity to S. aureus in milk. Different bacteria concentrations caused proportional decreases in the resonant frequency. The dissipation changes confirmed the sensitivity as they increased with increasing bacteria concentrations, indicating that S. aureus adsorbed to the crystal's surface by binding with the aptamer. As Figure 4 shows, the crystal experiences minimal frequency and dissipation shifts when exposed to milk without bacteria (8.9 ± 3.4 Hz and (2.1 ± 0.8) × 10 −6 frequency and dissipation shifts, respectively). Bacteria concentrations in milk were proportional to changes in the frequency and dissipation; increasing cell concentration caused decreasing frequency and increasing dissipation shifts.  Figure 5 illustrates the proportionality of the changes in frequency and dissipation due to increasing S. aureus concentrations in milk. The logarithmic trend indicates that quantification of the bacteria is possible, particularly from measuring frequency changes. The frequency and dissipation variations were calculated from the differences of the stable baselines (before and after sample exposure).   Figure 5 illustrates the proportionality of the changes in frequency and dissipation due to increasing S. aureus concentrations in milk. The logarithmic trend indicates that quantification of the bacteria is possible, particularly from measuring frequency changes. The frequency and dissipation variations were calculated from the differences of the stable baselines (before and after sample exposure). improves the layer's antifouling properties, as the neutral and polar surface is likely more effective for interacting with water molecules. Furthermore, the dithiol structure of DTTCOOH provides spacing for the water molecules to hydrogen bond with the layer, creating a thermodynamically favorable "water barrier" and largely preventing milk components from adsorbing. Compared to DTT, bare gold, MUA, and UDT crystals, the aptasensor's fouling significantly decreased by 82%, 94%, 95%, and 96%, respectively.

Sensing of Staphylococcus Aureus in Milk
The aptasensor showed high sensitivity to S. aureus in milk. Different bacteria concentrations caused proportional decreases in the resonant frequency. The dissipation changes confirmed the sensitivity as they increased with increasing bacteria concentrations, indicating that S. aureus adsorbed to the crystal's surface by binding with the aptamer. As Figure 4 shows, the crystal experiences minimal frequency and dissipation shifts when exposed to milk without bacteria (8.9 ± 3.4 Hz and (2.1 ± 0.8) × 10 −6 frequency and dissipation shifts, respectively). Bacteria concentrations in milk were proportional to changes in the frequency and dissipation; increasing cell concentration caused decreasing frequency and increasing dissipation shifts.  Figure 5 illustrates the proportionality of the changes in frequency and dissipation due to increasing S. aureus concentrations in milk. The logarithmic trend indicates that quantification of the bacteria is possible, particularly from measuring frequency changes. The frequency and dissipation variations were calculated from the differences of the stable baselines (before and after sample exposure).  For all concentrations of bacteria in milk tested, a change of approximately 8.9 Hz occurred, which is the mean blank. The standard deviation of the blank (SD blank ) is 3.4 Hz; therefore, the LOB = 14.5. The SD lc of the low concentration sample is 11.49 Hz; therefore, the LOD was found to be 33.4 CFU/mL. The sensor also showed a dynamic range of 10 2 to 10 6 CFU/mL, allowing it to quantify S. aureus in a wide range of concentrations.
According to the US Food and Drug Administration (FDA), a concentration of S. aureus greater than 10 4 CFU/mL is considered injurious to health [24]. As this amount is within the dynamic range of the developed sensor and well above the calculated limit of detection, this aptasensor is practical for monitoring milk contamination. The limit of quantification (LOQ) has been calculated according to Equation (4) as 101.2 CFU/mL. Bacteria-aptamer binding was analyzed by fitting the data to the Langmuir isotherm [25] (Figure 6): where the maximal frequency change is (∆f ) max , the dissociation constant is K d , and bacteria concentration is c. As the K d decreases, the binding strength of bacteria-aptamer increases. The calculated K d and (∆f ) max values for bacteria-aptamer binding in whole milk were found to be 270.9 ± 42.9 CFU/mL and 9.8 ± 3.5 kHz, respectively. For all concentrations of bacteria in milk tested, a change of approximately 8.9 Hz occurred, which is the mean blank. The standard deviation of the blank (SDblank) is 3.4 Hz; therefore, the LOB = 14.5. The SDlc of the low concentration sample is 11.49 Hz; therefore, the LOD was found to be 33.4 CFU/mL. The sensor also showed a dynamic range of 10 2 to 10 6 CFU/mL, allowing it to quantify S. aureus in a wide range of concentrations.
According to the US Food and Drug Administration (FDA), a concentration of S. aureus greater than 10 4 CFU/mL is considered injurious to health [24]. As this amount is within the dynamic range of the developed sensor and well above the calculated limit of detection, this aptasensor is practical for monitoring milk contamination. The limit of quantification (LOQ) has been calculated according to Equation (4) as 101.2 CFU/mL. Bacteria-aptamer binding was analyzed by fitting the data to the Langmuir isotherm [25] (Figure 6): where the maximal frequency change is (Δf)max, the dissociation constant is Kd, and bacteria concentration is c. As the Kd decreases, the binding strength of bacteria-aptamer increases. The calculated Kd and (Δf)max values for bacteria-aptamer binding in whole milk were found to be 270.9 ± 42.9 CFU/mL and 9.8 ± 3.5 kHz, respectively. Thus, the binding of bacteria to the aptamers resulted in a significant decrease in the frequency and increase in dissipation, which is evident of the viscosity contribution. In this case, the Sauerbrey equation cannot be directly applied for evaluation of the changes of the mass. However, certain rough estimation of the surface density of the aptamers and bacteria can be obtained. According to our data the changes of the frequency following immobilization of aptamers on the antifouling linker can be denoted by Δf = −21.51 ± 3.79 Hz, and corresponding changes in dissipation are ΔD = (3.94 ± 0.52) × 10 −6 . If we consider an aptamer molecular weight of about 18.6 kDa, we obtain the surface density of the nucleic acid, equivalent to about 4.8 × 10 12 molecules per cm 2 . This result is very similar to those that can be found in the literature for an aptamer monolayer specifically bonded to a surface [26].
The changes in frequency and dissipation following the addition of the S. aureus at concentration 10 7 CFU/mL are 139.6 ± 22.6 Hz and (13.2 ± 3.7) × 10 −6 , respectively. Then, the surface mass density of bacteria can be estimated as (0.96± 0.16) µg·cm −2 . Considering Thus, the binding of bacteria to the aptamers resulted in a significant decrease in the frequency and increase in dissipation, which is evident of the viscosity contribution. In this case, the Sauerbrey equation cannot be directly applied for evaluation of the changes of the mass. However, certain rough estimation of the surface density of the aptamers and bacteria can be obtained. According to our data the changes of the frequency following immobilization of aptamers on the antifouling linker can be denoted by ∆f = −21.51 ± 3.79 Hz, and corresponding changes in dissipation are ∆D = (3.94 ± 0.52) × 10 −6 . If we consider an aptamer molecular weight of about 18.6 kDa, we obtain the surface density of the nucleic acid, equivalent to about 4.8 × 10 12 molecules per cm 2 . This result is very similar to those that can be found in the literature for an aptamer monolayer specifically bonded to a surface [26].
The changes in frequency and dissipation following the addition of the S. aureus at concentration 10 7 CFU/mL are 139.6 ± 22.6 Hz and (13.2 ± 3.7) × 10 −6 , respectively. Then, the surface mass density of bacteria can be estimated as (0.96± 0.16) µg·cm −2 . Considering that S. aureus has a spherical shape of a diameter approximately 0.5 µm, and that the density of the cytoplasm is approximately 1 g·cm 3 [27], one can estimate that the average mass of one bacterium is 0.065 pg. Therefore, the surface density of the bacteria can be estimated as 1.48 × 10 7 bacteria·cm −2 . Considering that the cross-sectional area of one bacterium is approximately 0.2 µm 2 , at full coverage, the surface density will be 5 × 10 8 bacteria·cm −2 , which is much higher in comparison with those estimated from frequency changes. However, considering that S. aureus forms colonies, the surface density can be lower in comparison with those estimated from frequency changes. It is also evident that the surface density of bacteria is much lower than those of the aptamers. This means that a large number of aptamers bind to one bacterium.
In the literature, there have been a limited number of aptasensors for S. aureus explored. Table 2 summarizes some of the most sensitive aptasensors, which have LODs that are similar to this work's TSM-based aptasensor. However, some of the aptasensors were tested in culture, which is less fouling than whole milk. Our developed TSM aptasensor has excellent sensitivity that is better or comparable to other reported aptasensors. Moreover, our sensor has the advantage of operating in raw milk due to its antifouling thiol linker, DTT COOH , making detection rapid and efficient for the analysis of milk.

Effect of Aptamer/Ethanolamine Immobilization and Bacteria Binding on the Viscoelastic Properties of the Sensing Layers
It is possible to estimate the mass of aptamer immobilized on the electrode with the Sauerbrey equation, as well as ethanolamine (used to deactivate the activated carboxyl groups of the SAM and increase the antifouling characteristics of the surface). The mass of aptamer was found to be 30.0 ± 5.1 ng, while ethanolamine was 4.5 ± 2.9 ng. These values were then used for the subsequent calculations.
We also analyzed changes in the viscosity coefficient, η, shear modulus, µ, and penetration depth, Γ, following incubation of the DTT COOH SAM with aptamer, ethanolamine, and bacteria. Since it was not possible to measure the viscoelastic properties of the thiol layer (the functionalization of the thiols was conducted in vial, not in a flow), the values of the viscoelastic parameters are relative and not absolute. The viscoelastic parameters are shown in Table 3. The analyses were performed on three independent measurements and the data are represented as mean and standard deviation. The variation of the shear modulus, µ, for the aptamer layer has a magnitude that is comparable with biomolecules, and certainly smaller than the quartz crystal (~10 10 Pa) [37]. Regarding the viscosity coefficient, η, this was lower than compact protein layers such as those obtained with β-casein (~10 −4 Pa·s). This may suggest that the aptamer layer is not quite compact, but rather that the nucleic acid tends to have a loose, potentially globular three-dimensional structure which does not allow for high density packing. This would agree with the idea that aptamers need to fold in order to perform their receptor function. The penetration depth, Γ, of the mechanical evanescent wave is around 200 nm for a crystal with a fundamental frequency of 8 MHz [38]. This value varies substantially little following the formation of a compact SAM, while by immobilizing polymeric molecules, one should see a significant reduction. In fact, following the immobilization of the aptamer, the penetration depth is reduced by about 1/6, emphasizing how effectively the aptamer is present on the layer. However, due to its steric characteristics, the aptamer does not pack to form a dense layer. This is an important prerequisite for the formation of an aptasensor as the aptamer has greater freedom to rearrange itself in the presence of the specific analyte and bind more effectively to it.
The variation of the shear modulus for ethanolamine is less than that obtained following the incubation of the aptamer, meaning that ethanolamine is precisely bound to the SAM, contributing to the formation of a dissipative and non-elastic mix layer. However, being a small molecule, it has less influence on the elastic properties. In the case of the viscosity coefficient, η, this is also varied to a very small extent, indicating that this small molecule has very little influence on the density characteristics of the functionalized layer. However, as one would expect, the coefficient of viscosity increases slightly (the mix layer, with the contribution of ethanolamine, becomes more viscous). The penetration depth changed very little compared to the aptamer, confirming that it is precisely the ethanolamine that binds to the activated carboxyl groups of DTT COOH , which, being a small molecule, has little influence on the path of the evanescent acoustic wave. Additionally, the small variation in penetration depth also demonstrates that ethanolamine molecules bind distantly from each other (i.e., only where the DTT COOH tails are) and therefore not densely.
Following the incubation of the sensing layer with bacterial cells, the shear modulus increases to values greater than those of aptamer/ethanolamine alone, while the viscosity coefficient decreases. It is likely that the bacterial cell, by binding to the free end of the aptamers, reduces the movement of the latter, causing the formation of a more tightly packed layer and, consequently, one which is more elastic and slightly less dissipative (hence the increase in the shear modulus and the reduction of the coefficient of viscosity). However, the penetration depth does not vary significantly, increasing only slightly compared to the aptamer/ethanolamine heterolayer, since the acoustic wave propagates better in the presence of a more compact structure.

Specificity of Staphylococcus aureus Detection
To determine if the developed aptasensor is specific to S. aureus, it was also tested against 10 7 CFU/mL of P. aeruginosa and E. coli in whole milk. The frequency and dissipation were measured for each (Figure 7). We used relatively high concentrations of bacteria to be sure that no binding of bacteria other than S. aureus occurs, even at high concentrations.

Specificity of Staphylococcus aureus Detection
To determine if the developed aptasensor is specific to S. aureus, it was also tested against 10 7 CFU/mL of P. aeruginosa and E. coli in whole milk. The frequency and dissipation were measured for each (Figure 7). We used relatively high concentrations of bacteria to be sure that no binding of bacteria other than S. aureus occurs, even at high concentrations. The overall change in both frequency and dissipation are much higher for the aptasensor in response to S. aureus compared to the other bacteria. With S. aureus, the overall frequency variation was approximately 140 Hz, while E. coli was 27 Hz, and P. aeruginosa was only 16 Hz, representing an 80% and 89% greater signal for S. aureus compared to these other bacteria, respectively. Though some frequency change is observed for the other bacteria (due to a minimal fouling of milk), the much stronger signal for S. aureus shows that this sensor is still quite specific to the target bacteria and can be used to determine the specific contamination of samples caused by S. aureus.

Conclusions
A thickness shear mode (TSM) acoustic aptasensor was developed for detecting Staphylococcus aureus in whole UHT cow's milk. Detection was achieved without treating the milk due to the antifouling layer of the aptasensor, which employs the thiol linker 3dithiothreitol propanoic acid (DTTCOOH). We have shown that after linker synthesis, Fourier transform infrared spectroscopy (FTIR) can be used to analyze and confirm the structure of the desired molecule. Once DTTCOOH was linked to the aptamer, the antifouling The overall change in both frequency and dissipation are much higher for the aptasensor in response to S. aureus compared to the other bacteria. With S. aureus, the overall frequency variation was approximately 140 Hz, while E. coli was 27 Hz, and P. aeruginosa was only 16 Hz, representing an 80% and 89% greater signal for S. aureus compared to these other bacteria, respectively. Though some frequency change is observed for the other bacteria (due to a minimal fouling of milk), the much stronger signal for S. aureus shows that this sensor is still quite specific to the target bacteria and can be used to determine the specific contamination of samples caused by S. aureus.

Conclusions
A thickness shear mode (TSM) acoustic aptasensor was developed for detecting Staphylococcus aureus in whole UHT cow's milk. Detection was achieved without treating the milk due to the antifouling layer of the aptasensor, which employs the thiol linker 3-dithiothreitol propanoic acid (DTT COOH ). We have shown that after linker synthesis, Fourier transform infrared spectroscopy (FTIR) can be used to analyze and confirm the structure of the desired molecule. Once DTT COOH was linked to the aptamer, the antifouling character of the sensor significantly improved. Relative to the DTT COOH layer, the DTT COOH -aptamer layer experienced less milk fouling by approximately 88% and the frequency and dissipation variations were minimal in whole milk.
We used the frequency variation and dissipation data obtained during the construction of the aptasensor to demonstrate the correct immobilization of the components. In particular, the DNA aptamers bind to the surface in a non-dense manner, which favors recognition and binding with the analyte. Ethanolamine plays a marginal role in the variation of the viscoelastic parameters, which was expected, as it is a small molecule. However, the binding of bacteria to the sensing surface resulted in an increasing shear modulus, evidencing the reduction in the mobility of the aptamer layers.
The developed aptasensor achieved excellent sensitivity and specificity and was able to successfully detect and quantify S. aureus in milk. As the 33 CFU/mL limit of detection and 101 CFU/mL limit of quantification are significantly below the EU's safe limit for bacteria in milk products, the aptasensor is practical for rapid and sensitive detection in the milk industry. Additionally, the sensor was found to be specific to S. aureus compared to other tested bacteria. However, to make TSM more user-friendly, its design requires further engineering. The TSM aptasensor developed in this work demonstrates that a sensor based on the novel antifouling linker DTT COOH can rapidly detect and quantify S. aureus directly in raw whole UHT milk samples. Further work will explore the use of this sensor for different bacteria using their unique aptamers.