Apparatus and method for performing ligand binding assays on microarrays in multiwell plates
Ellipsometry systems for imaging binding events between analytes in a sample and an array of ligands in an evanescent field generated by a beam of light reflected from the plane of the ligands is adapted to a multiwell plate structure in common use. In one example, a film of prism arrays is affixed to the underside of the plate with each prism array located in registry with a well and ligand arrays being immobilized on the (planar) bottom surface of the wells. The prism array may be formed in a film and juxtaposed with the bottom surface of the plate or a prism array can be made integral with the plate bottom of a multiwell plate.
1. An apparatus, comprising:
a plate having therein a plurality of wells, each of the wells having a planar bottom surface for receiving a sample for analysis, wherein the plate has a transparent planar underside; and
an array of prisms affixed to the underside, a plurality of prisms of the array being in registry with a corresponding one of the plurality of wells.
2. The apparatus as in claim 1 , wherein the plurality of wells is arranged in rows and columns and wherein the array of prisms is formed as a continuous film.
3. The apparatus as in claim 2 , wherein the continuous film comprises a layer integral with the underside of the plate.
4. The apparatus as in claim 1 , wherein each prism of the array has a cross-sectional shape of a convex figure.
5. The apparatus of claim 4 , wherein the convex figure includes a trapezoid.
6. The apparatus of claim 5 , wherein a thickness t between the base of the plurality of prisms and the planar bottom surface of the corresponding one of the plurality of wells is given by
t
=
nP
cot
(
θ
)
2
,
wherein P is a pitch of the plurality of prisms, θ is an angle of incidence of a beam of light totally internally reflected from the planar bottom surface of the corresponding one of the plurality of wells, and n is an integer.
7. The apparatus as in claim 1 , wherein the array of prisms includes a uniform pattern of prisms formed in a sheet of transparent material.
8. The apparatus as in claim 7 , wherein the prism sheet has a corrugated surface on one side and a smooth surface on the other, wherein the corrugated surface includes a plurality of uniform linearly arrayed prism structures.
9. The apparatus as in claim 1 , wherein the array of prisms is integral with the underside of the plate.
10. The apparatus as in claim 1 , further comprising a top member against a top surface of a substrate, wherein the top member has an array of through apertures exposing portions of the top surface there.
11. The apparatus as in claim 10 , wherein a plurality of ligand arrays are immobilized on the top surface of a substrate at an exposed portion.
12. The apparatus of claim 1 , wherein a maximum height h of the plurality of prisms is given by
h
<
_
P
cot
(
θ
)
2
,
wherein P is a pitch of the plurality of prisms and θ is an angle of incidence of a beam of light totally internally reflected from the planar bottom surface of the corresponding one of the plurality of wells.
13. The apparatus of claim 1 , wherein a distance x between adjacent prisms of the plurality of prisms is given by
x
>
_
P
(
1
-
cot
2
(
θ
)
)
2
,
wherein P is a pitch of the plurality of prisms and θ is an angle of incidence of a beam of light totally internally reflected from the planar bottom surface of the corresponding one of the plurality of wells.
14. A system, comprising:
a plate having therein a plurality of wells, each of the wells having a planar bottom surface including an array of immobilized ligands to receive a sample for analysis,
wherein the plate has a transparent planar underside, and an array of prisms affixed to the underside, a plurality of prisms of the array being in registry with a corresponding one of the plurality of wells and with an associated array of immobilized ligands; and
a source of a beam of light configured to direct the beam to a selected area of the array of immobilized ligands in a manner to achieve total internal reflection and to generate an evanescent field in the plane of the associated array of immobilized ligands.
15. The system as in claim 14 , further comprising a sensor for sensing or measuring localized variations in the beam or beams reflected at the prisms.
16. The system as in claim 15 , wherein the sensor senses or measures localized changes in intensity indicative of phase changes between the horizontal and vertical polarization vectors of a polarized beam of light responsive to the occurrence of binding between analytes in a sample in a well and ligands in an array.
17. The system as in claim 14 , further comprising an optical system for directing the beam in a manner to achieve total internal reflection and to generate an evanescent field in the plane of the ligand array associated with a selected one of the prism array or position in the prism array.
18. The system as in claim 14 , further comprising a sensor for sensing or measuring localized variations in the reflected beam.
19. The system as in claim 14 , wherein the beam is a beam of polarized light.
20. A method of detecting binding events at an array of ligands immobilized on planar bottom surfaces of wells of a multiwell plate, the method comprising:
transmitting a beam of polarized light to a prism film affixed to an underside surface of the plate, the prism film including a plurality of prisms in registry with a corresponding one of the wells, the prism film being of a material and geometry to permit the beam of polarized light incident thereto to be totally internally reflected in a manner to generate an evanescent field in a plane of the array of ligands at which the beam is directed; and
obtaining an image of intensity variations in the reflected beam to detect binding events at said array of ligands.