Method for measuring analyte transport across cell membranes using X-ray fluorescence
View Patent ↗The present invention includes a method and apparatus for measuring the transport of an analyte through a cell membrane. One or more cells expressing a plurality of ion channels are provided. These cells are loaded with the analyte so that the cells contain at least 10 picograms of the analyte within a volume defined by the area of an x-ray excitation beam and a depth of five times the 1/e attenuation depth for at least one characteristic x-ray signal of the analyte as attenuated by water. The unloaded analyte is removed and the amount of the analyte in the cells is measured with x-ray fluorescence.
1. A method for measuring the transport of an analyte through a cell membrane, comprising the steps of providing one or more cells expressing a plurality of ion channels, loading the one or more cells with an analyte so that the cells comprise at least 10 picograms of the analyte within a volume defined by the area of an x-ray excitation beam and a depth of five times the 1/e attenuation depth for at least one characteristic x-ray signal of the analyte as attenuated by water, removing the unloaded analyte substantially from the volume, exciting the analyte with a polychromatic x-ray beam, and measuring the x-ray fluorescence of the analyte with an x-ray detector.
2. The method of claim 1 , further comprising unloading the cells and measuring the analyte after it is unloaded from the cells.
3. The method of claim 1 , further comprising lysing the cells prior to measuring the x-ray fluorescence.
4. The method of claim 1 , further comprising reducing matrix of the cells prior to measuring the x-ray fluorescence.
5. The method of claim 1 , further comprising drying the cells prior to measuring the x-ray fluorescence.
6. The method of claim 1 , wherein prior to loading the cells with the analyte, the analyte is substantially depleted in the volume defined by the area of the x-ray fluorescence excitation beam that is incident on the cells and a depth of five times the lie depth for at least one characteristic signal of the analyte in water.
7. The method of claim 1 , wherein the analyte comprises an element having an atomic number greater than 10.
8. The method of claim 1 , wherein the analyte comprises a chemical element selected from the group consisting of zinc, cadmium, thallium, sodium, potassium, rubidium, cesium, magnesium, calcium, barium, strontium, chlorine, bromine, and iodine.
9. The method of claim 1 , wherein the cells are retained in a chamber and wherein at least one portion of the chamber is translucent or transparent to x-rays.
10. The method of claim 9 , wherein the portion of the chamber that is translucent to x-rays passes at least 0.1% of the highest energy x-ray fluorescence signal that are emitted by the portion of the analyte that is located within 1 micron of the x-ray translucent location and that are normal to the x-ray translucent location and incident upon the x-ray translucent location.
11. The method of claim 1 , wherein the cells are immobilized such that a quantity of cells equivalent to at least 1% of the cells which are in the beam path of x-ray fluorescence excitation beam at the beginning of an x-ray fluorescence measurement are retained in the beam path of x-ray fluorescence excitation beam for a period of time which is greater than the measurement time of the x-ray fluorescence measurement.
12. The method of claim 1 , wherein the cells are immobilized such that a quantity of cells equivalent to at least 1% of the cells which are in the beam path of x-ray fluorescence excitation beam at the beginning of an x-ray fluorescence measurement are retained in the beam path of x-ray fluorescence excitation beam for at least 10 seconds.
13. The method of claim 1 , wherein the cells are retained in a chamber, and the orientation of the chamber and the x-ray excitation beam and the x-ray detector allows at least a portion of the population of cells to occupy the volume defined by the intersection of the x-ray excitation beam path and the viewable volume of the x-ray detector.
14. The method of claim 13 , wherein the portion of the population of cells comprises at least 100 picograms of the analyte occupying the volume defined by the intersection of the x-ray excitation beam path and the viewable volume of the x-ray detector.
15. The method of claim 14 , wherein the portion of the population of cells comprising at least 100 picograms of the analyte to occupy the volume defined by the intersection of the x-ray excitation beam path and the viewable volume of the x-ray detector, any barrier between the cells and the x-ray fluorescence detector attenuates the portion of the highest energy x-ray fluorescence signal and that is emitted normal to the barrier by the analyte that is located within 5 microns of the barrier by less than about 99.9% or allows at least 0.1% of the highest energy x-ray fluorescence signal from analyte to pass.