Portable high gain fluorescence detection system
View Patent ↗An instrument for fluorometric assays in liquid samples is disclosed. The instrument may include multiple optical channels for monitoring a first fluorophore associated with a target analyte and a second fluorophore associated with a control. The disclosed instrument finds utility in any number of applications, including microfluidic molecular biological assays based on PCR amplification of target nucleic acids and fluorometric assays in general.
1. An apparatus for performing a fluorescence assay on a sample contained between a pair of optical windows disposed on a common optical path through a microassay cartridge, the paired windows defining a detection chamber therebetween, the first window of the pair defining a first aspect of the microassay cartridge, the second window of the pair defining a second aspect of the microassay cartridge, the second window having a compliant, optically transparent and thermally conductive film sealed thereover, the apparatus comprising:
a) a housing adapted for receiving the microassay cartridge;
b) a scanning detector head with one or more optical channels, each optical channel having an excitation light source, an objective lens for directing the excitation light into the detection chamber and for receiving fluorescence emissions therefrom, and a emissions sensor for detecting the fluorescence emissions and outputting a voltage in response thereto, wherein the scanning detector head is enabled to scan in a series of spatially resolved steps across the detection chamber; and,
c) a variable temperature heating block mounted in the housing, the heating block having a top face that is optically flat and mirrored, wherein the mirrored face is configured to reflect fluorescent emissions exiting the second window back into the objective lens while controlling the temperature of the detection chamber, and wherein the heating block is rigidly mounted to a baseplate in the housing and a docking saddle is rigidly supported on the baseplate above the heating block, the docking saddle operably supporting a floating stage, the floating stage comprising a docking bay for receiving the removable cartridge and a spring suspension configured to contact the removable cartridge against the mirrored face of the heating block.
2. The apparatus of claim 1 , wherein the variable temperature heating block is configured with a heating element and a temperature monitor for controlling the temperature in the detection chamber when contacted thereto.
3. The apparatus of claim 1 , wherein the variable temperature heating block is enabled to melt and anneal a fluorescent probe complex in the detection chamber while quantitatively measuring fluorescence emissions of the probe.
4. The apparatus of claim 3 , wherein the apparatus is configured for performing a FRET melting temperature determination.
5. The apparatus of claim 1 , wherein the heating block is machined from aluminum and coated with an underlayer of copper under nickel before deposition of a chromium mirror face.
6. The apparatus of claim 5 , wherein the chromium mirror face is electropolished.
7. The apparatus of claim 3 , wherein the fluorescent probe complex comprises a molecular beacon.
8. The apparatus of claim 4 , wherein the fluorescence probe complex comprises a nucleic acid target of the assay, and the FRET melting curve is confirmatory for an identification of the assay target.
9. The apparatus of claim 1 , wherein the fluorescence assay is an assay for a nucleic acid target or a protein target in the sample.
10. The apparatus of claim 1 , wherein the floating stage with docking bay for receiving the removable cartridge is operated on by a clamping lever mounted on the docking saddle, the clamping lever operating to oppose the spring suspension when reversibly lifting the floating stage away from the heating block while loading or unloading the cartridge.
11. The apparatus of claim 10 , further comprising a pneumatic interface manifold formed within the baseplate, wherein the clamping lever operates to reversibly engage the removable cartridge with one or more pneumatic outlets defining a pneumatic interface array, each the pneumatic outlet operatively controlling a pneumatic subcircuit of the removable cartridge.
12. The apparatus of claim 11 , wherein the pneumatic interface array is configured to engage mating pneumatic inlets in the removable cartridge.
13. The apparatus of claim 12 , wherein a gasket is disposed on the removable cartridge, the gasket comprising a perforated elastomeric member for sealing the pneumatic interface array to the mating pneumatic inlets.
14. The apparatus of claim 1 , wherein the scanning detector head is moveably mounted on tracks affixed to the floating stage.
15. The apparatus of claim 14 , wherein the scanning detector head is operatively driven in a series of linear steps on the tracks while scanning across the detection chamber of the removable cartridge.
16. The apparatus of claim 1 , wherein the scanning detector head comprises an embedded microprocessor configured to enable scanning in spatially resolved steps along a sampling transect across the detection chamber, digitizing the emissions sensor voltages for each step, summing positive digital signals greater than a threshold value for each step, and outputting the sum.
17. The apparatus of claim 16 , wherein the positive digital signals are single-bit digital signals.
18. The apparatus of claim 16 , wherein the apparatus further comprises a programmable host controller configured for digitally receiving the sum from the embedded microprocessor and for calculating and displaying or transmitting a result of the fluorescence assay to a user.