A104 500

Carbon dots were synthesized using citric acid and β-alanine according to a previous report [8 (link)] with a slight modification, outlined in Figure 1. We used citric acid monohydrate (Fisher #A104-500, Pittsburg, PA, USA) as a carbon source instead of anhydrous citric acid (Fisher #BP339-500, Pittsburg, PA, USA) at the same molar ratio and observed similar results. Briefly, CDs were synthesized by mixing 1:2 molar ratio of citric acid and β-Alanine, where one gram of citric acid was mixed with 0.9 g of β-Alanine in 10 mL of distilled water (pH 3) in a conical flask. The mixture was homogenized using an Ultrasonicator (Ultrasonic Cleaner FS30, Fisher Scientific, Pittsburg, PA, USA) until completely dissolved and then heated for 3 min in a commercial microwave oven (Model#JES2251SJ02, GE Appliance, Louisville, KY, USA) at 70% power level to proceed to carbonization and surface passivation. The obtained brownish solid was then dissolved in 10 mL of distilled water (pH 3).

A 10 mM solution of citric acid (A104-500; Fisher Scientific, USA) containing 1 mM DSS reference standard was prepared, and 600 μL were added to a 5 mm NMR tube (Norell; Morganton, NC, USA). The pH of the solution was adjusted in-tube in ~0.25 pH increments by addition of 0.5–2 μL volumes of dilutions of concentrated NaOH and HCl and four rounds of inversion and vortex mixing. For each pH point, the pH was measured in-tube using a calibrated accumet AB150 pH meter (Fisher Scientific, USA), then a 1D noesypr1d spectrum was collected (DS = 2; NS = 16) on a 600 MHz Bruker magnet equipped with a 5 mm cryoprobe and an Avance III HD console at the University of Georgia NMR Facility. Data were phased and referenced to DSS in TopSpin (v3.5pl7; Bruker). Custom Matlab scripts were used to obtain the most upfield citrate peak position for each pH. A 3rd-order polynomial was fit to the positions (R² > 0.99) and used with the ridge belonging to the same peak to estimate the pH of each culture at each timepoint.