Measuring the Time-Evolution of Nanoscale Materials with Stopped-Flow and Small-Angle Neutron Scattering

Summary

This protocol presents the use of a stopped-flow sample environment to quickly mix multiple liquid solutions in situ during a small-angle neutron scattering measurement and to study kinetic processes on nanometer length scales and second time scales.

Abstract

This paper presents the use of a stopped-flow small-angle neutron-scattering (SANS) sample environment to quickly mix liquid samples and study nanoscale kinetic processes on time scales of seconds to minutes. The stopped-flow sample environment uses commercially available syringe pumps to mix the desired volumes of liquid samples that are then injected through a dynamic mixer into a quartz glass cell in approximately 1 s. Time-resolved SANS data acquisition is synced with the sample mixing to follow the evolution of the nanostructure in solution after mixing.

To make the most efficient use of neutron beam time, we use a series of flow selector valves to automatically load, rinse, and dry the cell between measurements, allowing for repeated data collection throughout multiple sample injections. Sample injections are repeated until sufficient neutron scattering statistics are collected. The mixing setup can be programmed to systematically vary conditions to measure the kinetics at different mixing ratios, sample concentrations, additive concentrations, and temperatures. The minimum sample volume required per injection is approximately 150 µL depending on the path length of the quartz cell.

Representative results using this stopped-flow sample environment are presented for rapid lipid exchange kinetics in the presence of an additive, cyclodextrin. The vesicles exchange outer-leaflet (exterior) lipids on the order of seconds and fully exchange both interior and exterior lipids within hours. Measuring lipid exchange kinetics requires in situ mixing to capture the faster (seconds) and slower (minutes) processes and extract the kinetic rate constants. The same sample environment can also be used to probe molecular exchange in other types of liquid samples such as lipid nanoparticles, proteins, surfactants, polymers, emulsions, or inorganic nanoparticles. Measuring the nanoscale structural transformations and kinetics of exchanging or reacting systems will provide new insights into processes that evolve at the nanoscale.

Introduction

Small-angle neutron scattering (SANS) provides a unique way to measure the sizes, shapes, interactions, and organization of various materials on length scales from ≈1 nm to ≈100 nm^1,2,3. Recent instruments, including VSANS (very small-angle neutron scattering) instruments with focusing mirrors, push the limits toward measuring even larger length scales up to ≈1000 nm^4,5. In general, the unique scattering contrast inherent to neutron scattering methods offers several advantages in measuring the time-evolution of nanosc....

Protocol

1. Set up and initiate the stopped-flow system.

1. Turn on all pump power supplies and dynamic mixers using the power switch.
2. Initiate all pumps and valves in the stopped-flow system control graphical user interface (GUI) by entering the device configuration path and using the commands bus=qmixbus.Bus(), bus.open(), bus.start(), pump=qmixpump.Pump(), pump.enable(), and valve=ViciMultiposSelector() (see example initiation code available in an online open-source repository⁶⁴).
3. Calibrate the pumps before attaching....

Results

The representative neutron data shown here measure lipid exchange kinetics in the presence of methyl-β-cyclodextrin (mβCD), an additive that catalyzes the lipid exchange between vesicles with the exchange rate (k_e)^66,67. Previous fluorescence studies have shown that k_e depends on the mβCD concentration, and the half-life of the exchange process is on the order of minutes⁶⁸. The present experiments use stopped-flow TR-.......

Discussion

The current procedure describes the mixing device and the steps to perform stopped-flow TR-SANS measurements. The device and protocol are optimized for low-viscosity liquid samples where the time scales of interest are ≈1 s to 5 min. For time scales greater than 5 min, manually mixing the samples and loading them into standard scattering cells may be easier and desirable, especially for high-viscosity samples, gels, or pastes. Accessing time scales less than 1 s requires a different mixing apparatus, lower total vo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dynamic mixer	Analytical Scientific Instruments	462-0150A	Magnetically coupled rotor, binary dynamic mixer assembly (ternary type available), 0.15 mL dead volume (larger dead volume available)
Fluoropolymer tubing	IDEX Health & Science	1507L	PFA Tubing Natural 1/16 inch OD x 0.040 inch ID x 50 ft
Fluoropolymer 1/4-28 flangeless fittings	IDEX Health & Science	XP-245	PFA flangeless fitting with ferrules, 1/4-28 threading, 1/16 inch OD tubing
Glass syringes	Hamilton Company	81660	Hamilton 1000 series syringes, 10 mL (81660), model 1010 C syr, 1/4"-28 thread termination, other volumes available
High-pressure flow selector valves	Vici Valco	C85X-1570EUTB	Vici 10 position selector valves, 15000 psi max, 0.25 mm bore, 1/16 inch OD tubing, 10-32 coned threaded ports, USB universal actuator
High-pressure switch valves	Vici Valco	C82X-1574EUHB	Vici 4 port switch valves, 15000 psi max, 0.25 mm bore, 1/16 inch OD tubing, 10-32 coned threaded ports, USB universal actuator
High-pressure syringes	Cetoni	A2019000358	3 mL stainless steel syringe, 510 bar max, 21 mL/min flow rate max
Low-pressure flow selector valves	Vici Valco	C25-3180EUHB	Vici 10 position selector valves, max 250 psi liquid, 0.75 mm bore, 1/16 inch OD tubing, 1/4-28 threaded ports, USB universal actuator
neMESYS high-pressure syringe pumps	Cetoni	A3921000103	Max force 2600 N
neMESYS mid-pressure syringe pumps	Cetoni	A3921000131	Max force 1000 N
Power supply	Cetoni	A3921000127	Base 600, supplies power for up to 4 high pressure pumps
Quartz flow-through sample cell	Starna Scientific	3-2.30-Q-1/TC	Quartz micro flow cells, 2 mm path length (1 mm available), 2 mm by 2 mm by 30 mm internal dimension
Quartz windows	Technical Glass Products	NA	GE 124 Clear fused quartz ground and polished plates, 11.75 inch by 23.75 inch by 0.375 inch thick
Stainless steel 10-32 coned compression fittings	IDEX Health & Science	U-321X, U-320X	316 stainless steel ferrule (U-321X) and nut (U-320X) -Valco type, 10-32 coned, for 1/16 inch OD stainless steel tubing
Stainless steel tubing	IDEX Health & Science	U-102	Stainless Steel Tubing 1/16 inch OD x 0.020 inch ID, 10 cm, various precut lengths available
Syringe pump control software	Cetoni	T6000000004	QmixElements software for nemesys pumps, QmixSDK software development kit
Thermoelectric air conditioner	EIC Solutions	AAC-140C-4XT-HC	Thermoelectric air conditioner mounted on insulated enclosure to control the pump, valve, mixer, and sample temperature
T-slot railing	McMaster-Carr	47065T103	Aluminum t-slotted railing (1.5 inch by 1.5 inch) cut to various lengths
Vapor locking bottle caps	Cole-Parmer	EW-12018-02	Four 304 SS port inserts, 1/4"-28 threads, GL45 bottle cap size, PTFE body, SS threads, PP collar