Quantifying picomoles of analyte from less than 100 live bacteria: A novel method with a buffering hydrogel as an electrochemical cell.

Microenvironmental changes in the chemical surrounding of bacterial cells might have a profound impact on the ecology of biofilms. However, quantifying total amount of picomoles of analyte from a miniscule number of bacteria is an analytical challenge. Here we provide a novel microliter volume hydrogel based electrochemical cell platform suitable of coulometrically measuring hydrogen peroxide (H2O2) produced by less than 100 cells of Streptococcus sanguinis, a relevant member of the healthy oral microbiome. A morpholine moiety was incorporated into the polymer structure of the hydrogel to create a controlled microenvironment at biological pH. We calculated the buffering capacity of this hydrogel as 0.257 ± 0.135 molHNO3molMEA×ΔpH over the pH range of 7.2-6.2 by using a novel method designed for buffering hydrogels. The H2O2 sensors coated in microliter volume of buffering hydrogel showed no change in sensitivity within the pH range of 7.0-3.0, allowing for H2O2 measurements of S. sanguinis independent of any acid they produce. The novel platform was able to measure down to 22.7 ± 3.5 pmol H2O2 produced by less than 100 bacterial cells, which would otherwise not be attainable in large solution-based assays. These findings indicate that this is a suitable platform for quantifying metabolites from sub-milligram biological samples and may even be suitable for direct analysis of raw biofilms samples with little to no sample pretreatment.

Real-Time Monitoring of Biofilm Formation Using a Noninvasive Impedance-Based Method.

Biofilms are complex three-dimensional microbial communities that adhere to a variety of surfaces and interact with their surroundings. Because of the dynamic nature of biofilm formation, establishing a uniform technique for quantifying and monitoring biofilm volume, shape, and features in real-time is challenging. Herein, we describe a noninvasive electrochemical impedance approach for real-time monitoring of dental plaque-derived multispecies biofilm growth on a range of substrates. A working equation relating electrochemical impedance to live biofilm volume has been developed that is applicable to all three surfaces examined, including glass, dental filling resin, and Ca2+-releasing resin composites. Impedance changes of 2.5, 35, 50, and 65% correlated to biofilm volumes of 0.10 ± 0.01, 16.9 ± 2.2, 29.7 ± 2.3, and 38.6 ± 2.8 µm3/µm2, respectively. We discovered that glass, dental filling resin, and Ca2+-releasing dental composites required approximately 3.5, 4.5, and 6 days, respectively, to achieve a 50% change in impedance. The local pH change at the biofilm-substrate interfaces also monitored with potentiometry pH microsensor, and pH change varied according to biofilm volume. This impedance-based technique can be a useful analytical method for monitoring the growth of biofilms on a variety of substrates in real-time. Therefore, this technique may be beneficial for examining antibacterial properties of novel biomaterials.

Calibration-free Solid-State Ion-Selective Electrode Based on a Polarized PEDOT/PEDOT-S-Doped Copolymer as Back Contact.

Solid-contact ion-selective electrodes (ISEs) have the inherent advantage of being miniaturized in addition to maintaining high selectivity and sensitivity of the ionophore-based ISE. The major disadvantage of ISEs is the necessity of performing a calibration curve (varying the intercept in the linear calibration curve equation) each time before running experiments, which limits their application as one-time disposable sensors or for use in remote water sample analysis. To overcome these challenges, we designed a unique back contact made of 3,4-ethylenedioxythiophene (EDOT) and 4-(2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl-methoxy)-1-butanesulfonic acid, sodium salt (EDOT-S). The calibration-free ISEs showed near Nernstian responses of 57.2 ± 0.2 mV/log [K+] and 28.5 ± 0.3 mV/log [Ca2+], while maintaining their respective selectivity against major interfering ions. The detection limits for Ca2+ and K+ ISEs were 0.45 ± 0.01 and 1.68 ± 0.18 µM, respectively. The charging cycles of the PEDOT: PEDOT-S back contact allowed us to fix the background potential at a desired fixed intercept value across different ionophores (K+, Ca2+). This protocol was used to determine the K+ and Ca2+ contents in creek water samples. The activity and concentration of [Ca2+] and [K+] in a local creek was found to be 257 ± 7.3 and 28.1 ± 1.1 µM, respectively.

Enantioselective Synthesis of (-)-Halenaquinone.

The efficient, 12-14 step (LLS) total synthesis of (-)-halenaquinone has been achieved. Key steps in the synthetic sequence include: (a) proline sulfonamide-catalyzed, Yamada-Otani reaction to establish the C6 all-carbon quaternary stereocenter, (b) multiple, novel palladium-mediated oxidative cyclizations to introduce the furan moiety, and (c) oxidative Bergman cyclization to form the final quinone ring.

Real-time monitoring of the pH microenvironment at the interface of multispecies biofilm and dental composites.

Bacterial-mediated local pH change plays an important role in altering the integrity of resin dental composite materials in a dynamic environment such as the oral cavity. To address this, we developed a 300-µm-diameter, flexible, solid-state potentiometric pH microsensor capable of detecting and quantifying the local pH microenvironment at the interface of multispecies biofilm and dental resin in real time over 10 days. We used fluorinated poly(3,4-ethylenedioxythiophene) as the back contact in our newly developed pH sensor, along with a PVC-based ion-selective membrane and PTFE-AF coating. The high temporal resolution pH data demonstrated pH changes from 7 to 6 and 7 to 5.8 for the first 2 days and then fluctuated between 6.5 to 6 and 6 to 5.5 for the remaining 8 days with the resin composite or glass slide substrate respectively. We could observe the fluctuations in pH mediated by lactic acid production within the biofilm and the re-establishment of pH back to 7. However, acid production started to overwhelm buffering capacity with the continuous feed of sucrose cycles and reduced the local pH nearer to 5.5. No such changes or fluctuations were observed above the biofilm, as the pH remained at 7.0 ± 0.2 for 10 days. The localized real-time monitoring of the pH within the biofilm showed that the pH shift underneath the biofilm could lead to damage to the underlying material and their interface but cannot be sensed external to the biofilm.