Electrochemical Sensors for Oral Biofilm-Biomaterials Interface Characterization: a review

Abstract

Important processes related to the interaction of the oral microbiome with the tooth surface happen directly at the interface. For example, the chemical microenvironment that exists at the interface of microbial biofilms and the native tooth structure is directly involved in caries development. Consequentially, a critical understanding of this interface and its chemical microenvironment would provide novel avenues in caries prevention, including secondary caries that often occurs at the interface of the dental biofilm, tooth structure and dental material. Electrochemical sensors are a unique quantitative tool and have the inherent advantages of miniaturization, stability, and selectivity. That makes the electrochemical sensors ideal tools for studying these critical biofilm microenvironments with high precision. This review highlights the development and applications of several novel electrochemical sensors such as pH, Ca²⁺, and hydrogen peroxide sensors as scanning electrochemical microscope (SECM) probes in addition to flexible pH wire sensors for real-time bacterial biofilm-dental surface and dental materials interface studies.

Electrochemical sensors can be used to conduct research on various aspects of oral microbiology and dental materials. For instance, they can determine the amount of microbes in a sample, the pH, the amount of oxygen, and the amount of metals. In addition, they can be used to track the progress of dental materials research toward the development of more efficient anticaries properties.

Applications of electrochemical sensors to the study of dental materials and biofilms.

In general, microbial colonization and proliferation occurs in a similar fashion on the tooth surface as well as dental materials (Kolenbrander et al., 2010) (Kuramitsu et al., 2007)(Chubukov et al., 2014) (Stacy et al., 2014). Biofilm formation can be categorized into four major steps. The first step involves bacterial adhesion to the substrate, the second step formation of bacterial microcolonies, the third step formation of a polysaccharide matrix that forms a three-dimensional biofilm structure, and the fourth step bacterial dispersion, which is the result of polysaccharide matrix-cleaving enzymes among other mechanisms. The microbial microenvironment is involved in each of these steps, in particular the critical first step of irreversible adsorption to any substrate, the essential step for biofilm formation. These initial attachment steps are defined by specific interactions of microbial cell surface adhesins with the substratum, which in case of the oral biofilm is the acquired enamel pellicle (AEP). AEP-microbial interactions are driven by the chemical environment and involve cations like Ca²⁺ and Mg²⁺, both detectable through electrochemical sensors as described below. It has become evident that bacteria are capable of sensing the microenvironment above the substrate as well as the substrate interface itself (Xu et al., 2014) (Kolenbrander et al., 2002). Thus, modulating the microenvironment to either promote or prevent attachment is a vital approach to develop new biomaterials that aide in the prevention of caries development. For example, biomaterials capable of releasing antimicrobial agents or molecules, including cations are currently being evaluated. However, their efficacy is commonly determined by a top-down approach in which the biofilm volume grown on the substrate of interest is measured with confocal laser scanning microscopy or other viability assays, and then the substrate with the lowest biofilm volume or most inactivated cells is chosen as the most effective antimicrobial substrate. Although this is a rapid and efficient method for identifying the most effective antimicrobial substrate, it omits the most fundamental question: why is one substrate more effective than others? This is an important question to ask, since most microbial cells are able to rapidly adapt to antimicrobial substances and tolerance (or resistance) will eventually develop. Therefore, a more defined analysis, including what happened at the biofilm-biomaterial interface, might provide additional information to outsmart the microbes, or at least prolong certain antimicrobial activities.

While certain antimicrobial activities are assessed by measuring the release of components into the surrounding environment, these assays are almost all bulk solution based. Consequently, this means that they can only be used to determine the number of antimicrobial agents released by any antibacterial surface at a given time, but not the spatial and temporal concentrations over time. Typical methods involve immersing substrates or materials in the appropriate buffer solution for 12 hours or longer and then collecting an aliquot for ICP-MS or HPLC-MS analysis. This approach has limitations in terms of estimating the number of antimicrobial agents released in real time and the blind spots associated with microenvironments above the substrates. Bacteria can sense only a few micrometers around themselves or in the microenvironment above the substrate (Pellegrino et al., 2022) (Thar & Kühl, 2003). Thus, in order to design a more effective antimicrobial substrate, one must first gain a better understanding of this microenvironment.

The greatest challenge is the absence of a quantitative analytical tool capable of detecting and characterizing the chemical microenvironment several micrometers above the substrate, in particular under realistic biofilm growth conditions. As discussed earlier, pH and H₂O₂ appeared to be relevant and important parameters in oral biofilm studies. These key parameters, especially local pH and H₂O₂ chemical profile, played an important role in controlling the balance between beneficial and pathogenic bacterial species distribution inside the oral microbiome. Similarly, Ca²⁺ is another important analyte to understand the biofilm-mediated corrosion process as well as dental materials-biofilm local interaction. We recently developed an electrochemical method for designing new dental composites by using scanning electrochemical microscopy (SECM) or a bottom-up chemical approach(Sheet et al., 2021) (Sheet & Koley, 2019) (Harris et al., 2016) (X. Liu et al., 2011). SECM is a surface probe technique in which the tip or the chemical probe is positioned at a known distance above the substrate at micrometer precision, without touching it, with the aid of an amperometry approach curve. The details about the working principle of SECM can be found in the reference (Bard & Mirkin, 2012) (Lee et al., 1990). Since the probe never touches the surface of the substrate, one can ideally use this method for any sensitive substrate such as biofilm, tissue, or mammalian cells, as well as new biomaterials(Stoica et al., 2008) (Bard et al., 2006) (Abucayon et al., 2014) (Castro et al., 2011) (Hu et al., 2007). Typically, a 25 μm diameter platinum microelectrode is used as a redox probe to perform the negative feedback approach curve and determine the probe-substrate distance. However, recent efforts have been made to develop a new type of sensor probe or SECM tip that can function as a redox probe, as well as other analytes-sensing probes. For example, Ummadi et al. (Ummadi et al., 2016) developed a novel carbon-based Ca²⁺-ion-selective microelectrode (μISE) (25–35 μm diameter) with an extremely fast response time (<0.5 s) that is highly selective against major interfering metal ions (Figure 1A–E). This dual-function Ca²⁺-μISE has the unique capabilities of performing an amperometric approach curve to determine the probe-substrate distance and can then be switched to potentiometric mode to quantitatively map the Ca²⁺ release profile in three-dimensional space above pure bioactive glass (BAG) substrates. The study reports that the Ca²⁺ concentration at a 20 μm distance above the pure BAG was 1.4 mM in the presence of artificial saliva of pH 4.5, while the bulk Ca²⁺ concentration remained constant at 700 μM. As expected, the study reported the bulk Ca²⁺ concentration measured over 4 h didn’t show any change in Ca²⁺ concentration in the presence of artificial saliva of pH 4.5, 6.05 and 7.2, which confirms the limitations of traditional assays.

Figure 1:

(A) Schematic representation of the (left) dual-electrode pH sensor (25 μm diameter Pt wire for each tip) and (right) 25–30 μm diameter carbon-based solid-state calcium ion-selective microelectrode. (B) SECM-BAG experimental setup. A 0.5 mm Pt wire and an Ag/AgCl were used as counter and reference electrodes, respectively (not shown). (C) Three-dimensional SECM image of calcium ion release from the BAG surface when exposed to artificial saliva (pH 4.5). The image was taken at a constant height of 200 μm from the BAG surface. (D) Three-dimensional SECM image of pH distribution above the BAG surface. pH imaging was performed over 1.6 mm of the exposed BAG surface (100 and 1,000 μm above) in artificial saliva at pH 4.5. (F) (left) Local chemical environment above BAG composites. Average change in Ca²⁺ concentration (relative to artificial saliva) 20 μm above each BAG-containing resin composition in the presence of artificial saliva at pH 4.5. (right) Average change in pH at 20 μm above each BAG-resin composition in the presence of artificial saliva at pH 4.5. Values are presented as mean ± SD. BAG, bioactive glass. Adapted from (Ummadi et al., 2016) (Aponso et al., 2019)

Later, Aponso et al. (Aponso et al., 2019) further extended the study to develop new effective dental materials, such as resin-BAG, capable of producing a chemical microenvironment similar to that of pure BAG composites. This chemical approach from the ground up is more efficient than rapid screening for dental composite material optimization. They used a carbon-based pH-μISE and Ca²⁺-μISE to quantitatively map the local pH and Ca²⁺ concentration across the entire composite, demonstrating that one can measure precise Ca²⁺ concentrations above the resin-BAG composites that bacteria will encounter when they approach the substrate, adhere, and subsequently develop the biofilm. The maximum Ca²⁺ concentration released at a distance of 20 μm above the resin-BAG composites (BAG particle size <38 μm) has been determined to be 200 μM Ca²⁺ in the presence of pH 4.5 artificial saliva (Figure 1F). However, no change in local pH was observed, as the BAG loading in the resin composites (particle size 150 to <5 μm with V_f=0.32) was insufficient to titrate the local buffer capacity. An interesting observation in this data set was that the maximum amount of Ca²⁺ released was from dental composites with a particle size of less than 38 μm, with no significant increase in Ca²⁺ release below that size. This result contradicts the popular belief that one is required to have nanoparticles to increase the effectiveness of metal ion release. The next pertinent question was whether the amount of Ca²⁺ released was sufficient to have an effect on biofilm growth. Surprisingly, the volume of Streptococcus mutans biofilm in the biofilm was reported to be significantly lower on the BAG dental composite (0.59 ±0.38) × 10⁷ μm³) in comparison to resin only substrate (1.29 ± 0.53) × 10⁷ μm³). Thus, it is safe to assume that <200 μM Ca²⁺ was sufficient to inhibit biofilm growth on the resin-BAG composite. It is obvious from this study that without a random screening of different compositions, SECM can play a significant role in quantifying the local chemical environment, which is necessary when designing an effective dental composite.

Earlier, the development and application of electrochemical microsensors for the purpose of designing and characterizing new dental composites through quantitative mappings of the local chemical environment, such as pH and Ca²⁺ was discussed. In addition, it was described how SECM could be an effective tool for quantifying the volume of live biofilms, which was used to determine the efficacy of the resin-BAG composites. The discussion will now transition to extending this technique to the study of chemical interactions between members of the oral microbiome.

Application of SECM to study the local bacterial metabolic interactions in real time.

Joshi et al. (Joshi, Sheet, et al., 2017) (Joshi, Kreth, et al., 2017) developed a pair of 25 μm diameter hydrogen peroxide (H₂O₂) and redox sensors that function as an SECM probe to investigate the chemical interactions between acid-producing Streptococcus mutans (Sm) and H₂O₂-producing S. gordonii (Sg). The sensors were made by etching one of the two platinum electrodes and packing it with ionic liquid and multi-walled carbon nanotubes decorated with platinum (Figure 2). This approach results in the sensors being extremely stable in complex experimental solutions, having a very fast response time and a very low detection limit of less than 0.2 μM. As a result, the sensors are suitable for studying live biofilms. The Sg biofilm produced 70 μM H₂O₂, whereas the Sm produced enough acid lactic acid to lower the local pH from 7.66 to 5.5. However, an interesting chemical interaction was observed when Sg was surrounded by Sm (see figure). Sm produced enough lactic acid to create an entirely different microenvironment above Sg, causing the Sg to behave unexpected. It was discovered that the amount of H₂O₂ produced was 10 μM rather than 70 μM. This means that the ability of Sg to produce H₂O₂ was significantly reduced by the immediate acidic microenvironment, making it less effective at controlling pathogenic Sm bacterial species. In addition, the SECM probe can also be modified for detecting and quantifying glucose consumed by S mutans. Jayathilake et al (Jayathilake & Koley, 2020) developed a new dual-tip glucose sensing scanning electrochemical microcopy (SECM) probe by covalently immobilizing the glucose oxidase (GOD) enzyme onto an ultramicroelectrode (UME). The GOD-based SECM tip showed a high current density of 94.44 μA· mM⁻¹·cm⁻² from 0.10 to 1.0 mM at 37 °C as a result of the synergetic effects of f-MWCNTs and ionic liquid. Because of the high selectivity of the sensor, the sensor was able to quantify glucose consumption in the presence of sucrose. Sm preferentially consumed sucrose in a mixed diet of both sucrose and glucose. Furthermore, using this unique fast-response (~2 s) glucose sensor, the distribution of the glucose consumption profile in the local environment of S. mutans biofilm was mapped. These findings provide insight into how the fast-growing Sm creates nutrient-depleted regions that affect the survival and metabolic behavior of other bacterial species within the oral biofilm. The detection limit of the new 25 μm diameter glucose sensor is 10.0 μM with a linear range up to 4.0 mM. This sensor was successfully used to quantify the rate of glucose consumption of Sm biofilms in the presence of sucrose. Even though the SECM studies reported here were on single or dual-species biofilm, current studies are underway in our laboratory to obtain chemical images using dental plaque-derived multispecies biofilm.

Figure 2:

Schematic representation of (A) the preparation of Pt-decorated multiwalled carbon nanotubes (Pt–MWCNTs) and (B) the dual (H₂O₂ sensor/Pt) SECM probe. The dual probe comprises one 25 μm Pt electrode etched and packed with Pt–MWCNTs–ionic liquid to make the H₂O₂ sensor and a second 25 μm Pt electrode used to fix the sensor–biofilm distance in SECM experiments. (C) Optical micrograph (top view) of the dual probe; the black color depicts the H₂O₂ sensor packed with catalysts, and the yellow disc represents the Pt probe. (D) Schematic of the S. mutans bacteria gel biofilm substrate and the pH microprobe used in SECM experiments. € Z-directional pH profile mapping from 50 μm above the S. mutans biofilm to 1000 μm in the bulk solution after addition of 30 mM sucrose in artificial saliva (pH 6.0) at 37 °C. (F) Metabolic activity of S. gordonii and S. mutans in a dual biofilm in artificial saliva. Schematic of the dual-bacteria gel biofilm substrate: S. gordonii surrounded by S. mutans (Sm) used in SECM experiments. The white dashed line represents the x-direction probe scan curve recorded with H₂O₂ and pH ultramicrosensors to measure the H₂O₂ and pH profile on the dual biofilm. (G) X-direction pH profile 150 μm above the dual-bacteria biofilm in the presence of glucose (G), sucrose (S), and glucose plus sucrose (G + S) at pH 6.0 and 7.2. Glucose and sucrose concentrations are 1 and 30 mM, respectively. (H) X-direction H₂O₂ profile 150 μm above the dual-bacteria biofilm in the presence of G and G + S at pH 6.0 and 7.2. (I) Z-direction H₂O₂ and pH profile from 50 μm above S. gordonii in the dual-bacteria biofilm to 1000 μm above in the bulk solution in the presence of G + S at pH 6.0 (solid lines) and 7.2 (dashed lines). Adapted from (Joshi, Sheet, et al., 2017) (Joshi, Kreth, et al., 2017)

Other examples of the usefulness to characterize biofilms include SECM mapping of the redox microenvironment above Pseudomonas aeruginosa biofilms, which is impossible to perform with any other technique (Koley et al., 2011). We discovered that P. aeruginosa is capable of establishing a reducing microenvironment by producing reduced pyocyanin, which ultimately aids the bacteria in reducing Fe(III) to Fe(II) and thus facilitate the iron acquisition, which may confer a competitive edge on the bacteria over other species.

So far, we have discussed the application of SECM in studying bacterial biofilms. However, numerous studies (Koley & Bard, 2010)(Koley & Bard, 2012)(Ummadi et al., 2015) (B. Liu et al., 2000)(Schulte et al., 2010) (Zhan et al., 2007) (Stoica et al., 2008) (Carano et al., 2003) in applying this SECM-based technique have been reported in studying mammalian cells too, which is beyond the scope of this review. However, one interesting study worth mentioning is the application of SECM to determine the permeability of live cancer cells or the rate at which extremely hydrophilic ionic compounds such as ferrocyanide molecules can pass through mammalian cell walls without causing cell death (Koley & Bard, 2010). It was found that the permeability of ferrocyanide molecules passing through the living cell membrane was reported to be 6.5 ± 2.0 ×10⁻⁶ m/s in the presence of 0.17 mM TX-100 surfactant. This highlight the potential use of SECM to study other oral relevant interfaces such as found between biofilm and oral epithelial cells which is involved in the development of periodontal disease.

Electrochemical sensors to study the biofilm-material interface at real time.

So far, we have seen how SECM can be used to quantitatively map the chemical microenvironment of any given surface, whether it is composed of dental composites or a living biofilm. However, as biofilms develop and begin to form a three-dimensional structure, the surface microenvironment may change, resulting in the formation of a chemical gradient of nutrients and metabolites. Probing the chemical microenvironment at the biofilm-substrate interface is even more challenging, owing to the complexity of the matrix and the difficulty of developing an analytical method capable of tracking the microenvironment while maintaining a high selectivity for the primary analytes. Nguyen et al.(Nguyen et al., 2022) developed a 300 μm diameter unique flexible pH wire sensor that is capable of measuring changes in local pH with high sensitivity for 14 days in a complex high-protein bacterial growth medium while maintaining the highest hydrogen ion selectivity. This enabled real-time monitoring of this highly dynamic interfacial chemical microenvironment as the dental plaque derived multispecies biofilm grew on various substrates. The pH difference between biofilms and glass substrates was 1.5 units or higher, or pH 7.0 to <5.5, whereas the pH difference between biofilms and resin substrates was maximum 1.0 units, or pH 7.0 to 6.0 (Figure 3). In addition, pH changes occurred only at the bottom of the biofilm or at the biofilm-substrate interface, while the bulk pH or distance away from the biofilm surface remained constant at pH 7.0. This could be due to the confined space at the bottom of the biofilm, where the lactic acid produced is sufficient to titrate the buffer but not the bulk pH. This environment is similar to that of the human oral cavity, where the pH at the interface between biofilm and teeth or dental composites may be significantly different, whereas saliva is extremely efficient at maintaining a pH of approximately 7.2 to 7.4. That is why it is critical to understand how the interfacial pH changes over time as biofilm grows on teeth, dental composites, or gum tissues, as each of these scenarios can result in a different type of oral disease, ranging from a cavity to filling material failure or from a secondary cavity to gum tissue inflammation.

Figure 3:

(Top) Schematic diagram of (A) incubator setup with sensors, (B) 300-μm flexible ion-selective electrode design, and (C) flexible pH sensors on top of the substrate embedded in the biofilm. (Bottom) Real-time monitoring of pH at the interface of multispecies biofilm and resin composite substrates at the left (A) and right (B) spots. The pH in bulk solution far away from the biofilm (C). Fluorescence image of multispecies biofilm growth for 10 days with sucrose feeding every 8 h and placement of pH sensors on the left (D) and right (E) location of the resin substrate. Adapted from (Nguyen et al., 2022).

CONCLUSION

It is clear that electrochemical sensors have the potential to play a significant role in detecting and quantifying a diverse array of important metabolites produced by bacterial biofilms at high spatial and temporal resolutions. This capability can thereby assist microbiologists in elucidating various oral diseases and assist dental material scientists in developing more effective dental materials with increased stability and longevity. As demonstrated in the discussion, electrochemical sensors are capable of producing highly quantitative measurements and thus may aid in the development of more quantitative and standardized testing tools for new dental materials.