Sample manipulation and assay with rapid temperature change
AssigneeEssenlix Corporation
InventorsStephen CHOU, Wei DING, Ji QI, Hua Tan, Yufan Zhang
Life of this patent
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Abstract
Among other things, the present invention is related to devices and methods of performing biological and chemical assays, particularly an easy sample manipulation and/or a rapid change or a rapid thermal cycling of a sample temperature is needed (e.g. Polymerase Chain Reaction (PCR) for amplifying nucleic acids).
Claims54
A device for fluidically isolating a portion of a sample, comprising:
- a first plate, a second plate, a plurality of spacers, and a clamp, wherein:
- i. the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration, wherein one or both of the plates is flexible, wherein each of the plates comprises, on its respective surface, a sample contact area for contacting a fluidic sample, and wherein at a closed configuration the plates sandwich a sample to be analyzed into a layer, that has a thickness 200 um or less, and has an sample area at last 100 times larger than the sample thickness;
- ii. the plurality of spacers have a predetermined substantially uniform height that is equal to or less than 200 microns, wherein at least one of the spacers is inside the sample contact area;
- iii. the clamp has two operation modes:
- (a) a non-active mode, wherein the top ring and the bottom ring of the clamp do not push the first plate and second plate together;
- and
- (b) an active mode, wherein the top ring and the bottom ring of the clamp are configured to exert a force to (i) squeeze the first plate and the second plate and deform the area of the flexible plates that is under the compression of the clamp, and (ii) deform the spacers that are under the compression of the clamp and crush at least one of the spacers, thereby reducing the spacing between the two plates in that area, and wherein the reduction of the plate spacing reduces or prevents a fluidic flow between a sample portion encircled by the rings and a sample portion outside the rings,
- wherein in the open configuration the two plates are partially or completely separated apart, the spacing between the plates is not regulated by the spacers, and the clamp is in non-active mode, and the sample is deposited on one or both of the plates;
- and
- wherein in the closed configuration, at least a part of the sample deposited in the open configuration is compressed by the two plates into a layer of substantially uniform thickness, wherein the uniform thickness of the layer is regulated by the plates and the spacers.
A device for fluidically isolating a portion of a sample and providing a rapid temperature change in the portion, comprising:
- a first plate, a second plate, a plurality of spacers, and a clamp, wherein:
- i. the first plate and the second plate are movable relative to each other into different configurations, including an open configuration and a closed configuration, wherein one or both of the plates is flexible, wherein at least on plate has a thickness of 300 um or less, wherein each of the plates comprises, on its respective surface, a sample contact area for contacting a fluidic sample, and wherein at a closed configuration the plates sandwich a sample to be analyzed into a layer that has a thickness 200 um or less, and layer has an sample area at last 100 times larger than the sample thickness;
- ii. the plurality of spacers have a predetermined substantially uniform height that is equal to or less than 200 microns, wherein at least one of the spacers is inside the sample contact area;
- iii. the plates have a thin thickness that are configured to rapidly change the temperature of the sample;
- and
- iv. the clamp has two operation modes:
- (a) a non-active mode, wherein the top ring and the bottom ring of the clamp do not push the first plate and second plate together;
- and
- (b) an active mode, wherein the top ring and the bottom ring of the clamp apply a force to (i) squeeze the first plate and the second plate and deform the area of the flexible plates that is under the compression of the clamp, and (ii) deform the spacers that are under the compression of the clamp and crush at least one of the spacers, thereby reducing the spacing between the two plates in that area, and wherein the reduction of the plate spacing reduces or prevents a fluidic flow between a sample portion encircled by the rings and a sample portion outside the rings,
- wherein in the open configuration the two plates are partially or completely separated apart, the spacing between the plates is not regulated by the spacers, the clamp is in non-active mode, and the sample is deposited on one or both of the plates;
- and
- wherein in the closed configuration, at least a part of the sample deposited in the open configuration is compressed by the two plates into a layer of substantially uniform thickness, wherein the uniform thickness of the layer is regulated by the plates and the spacers.
The device of any prior claim, wherein the first plate or the second plate have a thickness less than or equal to 100 μm.
The device of any prior claim, wherein both the first plate and the second plate have a thickness less than or equal to 100 μm.
The device of any prior claim, further comprises a heating/cooling layer disposed on the exterior of either the first plate or the second plate, wherein the heating/cooling layer is configured to receive electromagnetic radiation.
The device of any prior claim, wherein the heating/cooling layer disposed on the exterior of either the first plate or the second plate, the heating/cooling layer has a conductance of 6×10−5 W/K or higher.
The device of any prior claim, wherein the heating/cooling layer disposed on the exterior of either the first plate or the second plate, the heating/cooling layer has a conductance of from 6×10−5 W/K to 1.5×10−4 W/K.
A device for rapidly changing the temperature of a thin fluidic sample layer with image monitoring, comprising:
- i. any prior device;
- ii. a heater, and
- iii. an imager;
- wherein the imager images an area of the sample and a portion of the plates in the area, and the imager is configured to characterize a process of a rapid temperature change of the sample.
A device for qPRC, comprising:
- i. any prior device;
- ii. a heater;
- and
- iii. an imager for detecting a label;
- wherein the imager images an area of the sample and a portion of the plates in the area.
The device of any prior claim, wherein the ring is circular, oval, rectangular, pentagonal, hexagonal, square, star, or any combination thereof with optional rounded corners.
The device of any prior claim, wherein the plates and the clamp are configured to change the temperature of the sample at a rate of 10° C./s or higher.
The device of any prior claim, wherein activation of the clamp exerts a pressure of 0.01 kg/cm{circumflex over ( )}2 or higher on the plates.
The device of any prior claim, wherein the clamp comprises a top ring on the first plate and a bottom ring on the second plate that are movable toward each other.
The device of any prior claim, wherein the first and/or second plate comprises a well and in the closed configuration the flow of sample from the inside to the outside of the well during thermal cycling or temperature changing is reduced compared to without using a clamping structure.
The device of any prior claim, wherein the plurality of clamps, each reduce the flow of the sample from the inside to the outside of the ring area during thermal cycling or temperature changing.
The device of any prior claim, wherein the plurality of clamps comprises at least 2, at least 4, at least 8, at least 16, at least 32 or at least 64 or more, top and bottom clamp rings, where the top rings are movable relative to the bottom rings, and when the plates are in the closed position the rings oppose each other and pinch different areas of the device to produce multiple isolated reaction chambers.
The device of any prior claim, wherein the clamp ring has a cross-section that is round, square, triangular, or rectangular with optional rounded corners.
The device of any prior claim, where the cross-section of the top ring has a shape that is different to the bottom ring.
The device of any prior claim, wherein in the active mode the clamp crushes the spacers between the rings, thereby creating an at least partially isolated reaction chamber in the device.
The device of any prior claim, wherein the ring area is less than 5 mm2, less than 2 mm2, less than 1 mm2, less than 0.5 mm2, or less than 0.1 mm2.
The device of any prior claim, wherein only one of the plates is flexible.
The device of any prior claim, wherein the clamp reduces air bubbles in the sample during thermal cycling or temperature changing compared to without using a clamping structure.
The device of any prior claim, wherein the sample in the clamped area has a more even temperature compared to without using a clamp.
The device of any prior claim, a fourth power of the inter-spacer-distance (ISD) of the spacers divided by the thickness (h) and the Young's modulus (E) of the plate (ISD4/(hE)) is 5×106 um3/GPa or less.
The device of any prior claim, wherein a product of the pillar contact filling factor and the Young's modulus of the spacers is 2 MPa or larger, wherein the pillar contact filling factor is the ratio of the contact area between spacer and the plate to the total plate area.
A method for rapidly changing the temperature of a fluidic sample, comprising:
- (a) depositing a sample onto the sample contact area of at least one of the plates of the device of any prior claim while in the open configuration;
- (b) closing the plates into the closed configuration and placing the clamp in the active mode;
- and
- (c) rapidly changing the temperature of the sample portion encircled by the rings.
The method of any prior method claim, wherein the sample is a reaction mixture.
The method of any prior method claim, wherein the sample comprises dNTPs, a thermostable polymerase, a template, and primers.
The method of any prior method claim, comprising thermocycling the sample portion encircled by the rings.
The method of any prior method claim, wherein the method comprises thermocycling the sample portion encircled by the rings through a plurality of cycles that comprise increasing the temperature of the reaction mix to a temperature of at least 90° C. and then decreasing the temperature of the reaction mix to one or more temperatures in the range of 40° C. to 80° C.
The method of any prior method claim, wherein the method comprises thermocycling the sample portion encircled by the rings through a plurality of cycles that comprise increasing the temperature of the reaction mix to a temperature of at least 90° C. and then decreasing the temperature of the reaction mix to one or more temperatures in the range of 40° C. to 65° C., then increasing the temperature of the reaction mix to one or more temperatures in the range of 60° C. to 65° C.
The method of any prior method claim, wherein the sample portion encircled by the rings is thermocycled through 10 to 50 cycles.
A method for performing quantitative real-time PCR (qPCR), comprising:
- (a) thermocycling a reaction mixture using a device of any prior device claim, wherein the reaction mixture comprises a pair of PCR primers, a polymerase, a fluorescence-quencher probe oligonucleotide, dNTPs, and a template;
- and
- (b) in each cycle, measuring a fluorescent signal generated by cleavage of a label from the fluorescence-quencher probe oligonucleotide.
The method of any prior qPCR claim, wherein the measuring is done by imaging the sample
The method of any prior qPCR claim, wherein the thermocycling is done by activating a heat source configured to radiate electromagnetic radiation towards a heating layer located on either the first plate or the second plate of the device.
The method of any prior qPCR claim, wherein thermocycling comprises a plurality of cycles that comprise increasing the temperature of the reaction mixture to a temperature of at least 90° C. and then decreasing the temperature of the reaction mix to one or more temperatures in the range of 40° C. to 80° C.
The method of any prior qPCR claim, wherein thermocycling comprises a plurality of cycles that comprise increasing the temperature of the reaction mix to a temperature of at least 90° C. and then decreasing the temperature of the reaction mix to one or more temperatures in the range of 40° C. to 65° C., then increasing the temperature of the reaction mix to one or more temperatures in the range of 60° C. to 65° C.
The method of any prior pPCR claim, wherein the sample is thermocycled through 10 to 50 cycles.
The method of any prior pPCR claim, wherein cleavage of the sample is measured by detecting fluorescence of the reaction mixture during at least some of the cycles.
The method of any prior qPCR claim, wherein the fluorescence-quencher probe oligonucleotide comprises a fluorophore and a quencher, and the polymerase cleaves the fluorophore or quencher from the fluorescence-quencher probe oligonucleotide, thereby generating a fluorescent signal.
The method of any prior qPCR claim, further comprising plotting the fluorescent signal over time.
The method of any prior qPCR claim, further comprising estimating the amount of the template in the sample using the plotted signal.
A method for performing digital PCR (dPCR), comprising:
- (a) thermocycling an emulsion using a device of any prior device claim, wherein each droplet of the emulsion comprises a reaction mix comprising a pair of PCR primers, a polymerase, a fluorescence-quencher probe, and dNTPs and wherein only some of the droplets comprise a template;
- and
- (b) determining which droplets fluoresce, wherein the fluorescence is generated by cleavage of a label from the fluorescence-quencher probe oligonucleotide by the polymerase.
The method of any prior dPCR claim, comprising determining the number of droplets that fluoresce.
The method of any prior dPCR claim, further comprising estimating the number of template molecules in the sample based on the number of droplets that fluoresce.
The method of any prior dPCR claim, wherein the polymerase is a DNA polymerase.
The device and method of any prior claim, wherein the sample contains or is suspected contain an analyte, wherein the analyte is nucleic acid.
The device and method of any prior claim, wherein the sample contains or is suspected contain an analyte, wherein the analyte is RNA.
The device, kit, system, or method of any prior embodiments, wherein the pressure provided by clamp on the card is 5 PSI, 10 PSI, 30 PSI, 60 PSI, 90 PSI, 100 PSI, 150 PSI, 200 PSI, 500 PSI 1000pSI, or in a range between any of the two values.
The device, kit, system, or method of any prior embodiments, wherein the spacers are arranged on the respective plates at a surface density of greater than one per um2, greater than one per 10 um2, greater than one per 100 35 um2, greater than one per 500 um2, greater than one per 1,000 um2, greater than one per 5,000 um2, or in a range between any of the two values.
The device of any prior embodiments, further comprising a hinge that connects the first plate and the second plate, and is configured to allow the two plates to rotate around 5 the hinge into different configurations.
Dependencies are inferred from the claim text.