Monitoring Enzymatic Reactions in Real Time Using Venturi Easy Ambient Sonic-Spray Ionization Mass Spectrometry

Abstract

We developed a technique to monitor spatially confined surface reactions with mass spectrometry under ambient conditions, without the need for voltage or organic solvents. Fused-silica capillaries immersed in an aqueous solution, positioned in close proximity to each other and the functionalized surface, created a laminar flow junction with a resulting reaction volume of ∼5 pL. The setup was operated with a syringe pump, delivering reagents to the surface through a fused-silica capillary. The other fused-silica capillary was connected to a Venturi easy ambient sonic-spray ionization source, sampling the resulting analytes at a slightly higher flow rate compared to the feeding capillary. The combined effects of the inflow and outflow maintains a chemical microenvironment, where the rate of advective transport overcomes diffusion. We show proof-of-concept where acetylcholinesterase was immobilized on an organosiloxane polymer through electrostatic interactions. The hydrolysis of acetylcholine by acetylcholinesterase into choline was monitored in real-time for a range of acetylcholine concentrations, fused-silica capillary geometries, and operating flow rates. Higher reaction rates and conversion yields were observed with increasing acetylcholine concentrations, as would be expected.

Figure 1

Schematic view of the V-EASI MS setup used for real-time measurements of surface-reactions.

Figure 2

Fluid dynamics modeling of the liquid junction (in the absence of enzymes on the surface) where an analyte was perfused from the left side at 0.5 μL/min and collected at the right side at 0.6 μL/min. The scale bar indicates sizes within the colored plane.

Scheme 1. Hydrolysis of Acetylcholine by Acetylcholinesterase (AChE) Yields Choline and Acetate

Figure 3

Representative traces of real-time mass spectrometry monitoring conversion of acetylcholine into choline by acetylcholinesterase, surface-bound to an organosiloxane polymer. Use of a small capillary inner diameter (o.d. 150 μm, i.d. 50 μm) and low flow rate (q_in = 0.5 μL/min, q_out = 0.6 μL/min) allows for a high yield of conversion. The plots show extracted ion chromatograms for acetylcholine (ACh, m/z 146.1176) and choline (Ch, m/z 104.1070). A steady-state condition was first established outside the enzyme surface, followed by a rapid movement (∼100 ms) into the enzyme surface, which caused a perturbation of the reaction conditions, shortly followed by a new steady-state condition. Moving out from the enzyme surface restored the reaction to its initial conditions.

Figure 4

Representative traces of real-time mass spectrometry monitoring conversion of acetylcholine (ACh, m/z 146.1176) into choline (Ch, m/z 104.1070) by acetylcholinesterase. Use of a large capillary inner diameter (o.d. 350 μm, i.d. 200 μm) and higher flow rate (q_in = 8 μL/min, q_out = 10 μL/min) leads to a low surface-to-volume ratio in combination with less time for substrate diffusion, resulting in lower conversion compared to what is obtained with smaller capillaries and lower flow rates.