WO2021189065A1 - Supports d'échantillons multi-sites, ensembles de station de pcr et procédés de fonctionnement de systèmes d'essai de pcr - Google Patents

Supports d'échantillons multi-sites, ensembles de station de pcr et procédés de fonctionnement de systèmes d'essai de pcr Download PDF

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Publication number
WO2021189065A1
WO2021189065A1 PCT/US2021/070133 US2021070133W WO2021189065A1 WO 2021189065 A1 WO2021189065 A1 WO 2021189065A1 US 2021070133 W US2021070133 W US 2021070133W WO 2021189065 A1 WO2021189065 A1 WO 2021189065A1
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WO
WIPO (PCT)
Prior art keywords
sample holder
pcr
site sample
channel
inlet
Prior art date
Application number
PCT/US2021/070133
Other languages
English (en)
Inventor
Paul Patt
Daniel Chu
Original Assignee
Siemens Healthcare Diagnostics Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Siemens Healthcare Diagnostics Inc. filed Critical Siemens Healthcare Diagnostics Inc.
Publication of WO2021189065A1 publication Critical patent/WO2021189065A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements

Definitions

  • the present disclosure relates to multi-site sample holders and methods used in automated polymerase chain reaction (PCR) testing of DNA/RNA templates extracted from a biological sample.
  • PCR polymerase chain reaction
  • Testing within diagnostic laboratories may involve extracting and quantifying one or more constituents in a biological sample obtained from a patient, such as from blood serum, blood plasma, urine, and the like.
  • PCR testing is a technique used to amplify a targeted DNA or RNA sequence from a few extracted DNA or RNA fragments (hereinafter the "DNA/RNA template") that have been extracted from the biological sample to millions or billions of copies within a short period of time.
  • DNA/RNA template a DNA or RNA fragments that have been extracted from the biological sample to millions or billions of copies within a short period of time.
  • PCR testing can be used for identification of DNA or RNA sequences involved in cancer or genetic disorders, such as cystic fibrosis, or for the identification and diagnosis of diseases caused by fungi, bacteria, and viruses.
  • PCR processing cycles of heating and cooling are repeated many times on a PCR solution containing DNA/RNA templates that have been extracted via sample processing.
  • PCR solution can include the DNA/RNA templates, a master mix and a primer or probe, and possibly a reagent or enzyme and possibly deionized water, leading to a large number of exact copies of the originally-extracted DNA/RNA templates.
  • an optical technique such as fluorescence staining may be used to determine the amount of replicated DNA/RNA that is present as well as provide for further DNA/RNA sequence analysis.
  • a multi-site sample holder includes a body having a top surface and a bottom surface, the body further comprising: an inlet groove formed into the bottom surface, an outlet groove formed into the bottom surface alongside the inlet groove, a detection recess formed into the bottom surface and connected to the inlet groove and the outlet groove, a fill port interconnected to at least the inlet groove; and a cover connected to the bottom surface wherein the cover interfaces with the body to form an inlet channel interconnected to the fill port, a detection region interconnected to the inlet channel, and an outlet channel, wherein multiple sites are formed in the detection region.
  • a PCR station assembly in another aspect, includes a base configured to receive a multi-site sample holder as described in claim 1; a clamp member configured to secure the multi-site sample holder to the base including a plurality of viewing apertures corresponding to each of the multiple sites; and a temperature-controlling element operable to heat and cool the base in intimate thermal contact with the multi-site sample holder.
  • a method of operating a PCR testing system includes providing a PCR station comprising a base, a clamp member, and a temperature-controlling element thermally coupled to the base; securing a multi-site sample holder between the base and clamp member, the multi-site sample holder including an inlet channel interconnected to a fill port, a detection region interconnected to the inlet channel including multiple sites, and an outlet channel interconnected to the detection region; inserting a volume of PCR solution into the fill port thus filling the detection region and the multiple sites; subjecting the PCR solution to heating and cooling by subjecting the base to cycles of heating and cooling with the temperature-controlling element to replicate tagged DNA/RNA templates to produce tagged replicated DNA/RNA; and after a predetermined number of heating and cooling cycles, measuring fluorescent emissions from the multiple sites in the detecting region with an optical interrogation apparatus by separately exciting each of the multiple sites including the tagged replicated DNA/RNA with a particular wavelength of light.
  • FIG. 1A illustrates a top perspective view of a multi-site sample holder according to one or more embodiments of the disclosure.
  • FIG. IB illustrates a cross-sectioned side view of a multi-site sample holder taken along section line 1B-1B of FIG. 1A according to one or more embodiments of the disclosure.
  • FIG. 1C illustrates another cross-sectioned side view of a multi-site sample holder taken along section line 1C-1C of FIG. 1A according to one or more embodiments of the disclosure.
  • FIG. ID illustrates a bottom perspective view of a multi-site sample holder, with the cover removed for illustration purposes, according to one or more embodiments of the disclosure.
  • FIG. IE illustrates a bottom plan view of a cover according to one or more embodiments of the disclosure.
  • FIG. IF illustrates a top plan view of a multi-site sample holder with the configuration of an example flow path and multiple detection sites being illustrated (shown dotted) according to one or more embodiments of the disclosure.
  • FIG. 1G illustrates a cutout view of the respective flow path for a PCR solution through a multi-site sample holder (flow path shown in isolation for illustration purposes) according to one or more embodiments of the disclosure.
  • FIG. 2A illustrates a perspective view of a PCR station assembly holding a multi-site sample holder according to one or more embodiments of the disclosure.
  • FIG. 2B illustrates a side view of a PCR station assembly holding a multi-site sample holder according to one or more embodiments of the disclosure.
  • FIGs. 2C-2D illustrate top and bottom perspective views, respectively, of a clamp member of a PCR station assembly according to one or more embodiments of the disclosure.
  • FIG. 2E illustrates a top perspective view of a base of a PCR station assembly including a pocket configured to receive a multi-site sample holder assembly according to one or more embodiments of the disclosure.
  • FIG. 2F illustrates a top perspective view of a temperature-controlling element operable to heat and cool the base of a PCR station assembly that is in intimate thermal contact with the multi-site sample holder according to one or more embodiments of the disclosure.
  • FIG. 3 illustrates a block diagram of a PCR testing assembly made up of a PCR station assembly holding a multi site sample holder that is being interrogated by an optical interrogation apparatus according to one or more embodiments of the disclosure.
  • FIG. 4 is a flowchart illustrating a method of operating a PCR testing assembly including a multi-site sample holder according to one or more embodiments of the disclosure.
  • FIG. 5 illustrates a partially-sectioned top view of an alternate embodiment of multi-site sample holder with multiple sites arranged around a plenum according to one or more embodiments of the disclosure.
  • FIG. 6 illustrates a partially-sectioned top view of an alternate embodiment of multi-site sample holder with multiple sites formed as a honeycomb according to one or more embodiments of the disclosure.
  • FIG. 7 illustrates a partial, cross-sectioned side view of a portion of the multi-site sample holder of FIG. 6 illustrating an example construction of the multiple sites according to one or more embodiments of the disclosure.
  • FIG. 8 illustrates a top plan view of a multi-site sample holder illustrating the alternate inlet and outlet port as well as an optional inclusion of grasping features enabling grasping with a robotic gripper according to one or more embodiments of the disclosure.
  • the present disclosure is directed at a multi-site sample holders for use in, for example, PCR testing.
  • the multi-site sample holders can provide a low- cost design that can be used with automated PCR processing for both amplification and detection phases of PCR processing.
  • the multi-site feature allows for the detection at multiple sites wherein a different master mix or chemistry can be provided at the different sites with different detection targets at each, for example.
  • the multi-site sample holder can be intended for a single use, and disposed thereafter.
  • the multi-site sample holder may be washed and reused.
  • the multi-site sample holder is miniaturized in that the multi-site sample holder can perform detection (e.g., fluorescence or other detection) on very small volumes of the PCR solution containing the extracted DNA/RNA templates, such as using volumes of PCR solution of less than or equal to 20mR, or even from 3mR to 20mR.
  • detection e.g., fluorescence or other detection
  • the PCR station assembly is provided that is configured to hold the multi-site sample holder in a defined position for the amplification and detection phases of PCR processing.
  • the multiple sites are subjected to multiple heating and cooling cycles to replicate the DNA/RNA templates.
  • the PCR station assembly holds the multi-site sample holder containing the replicated DNA/RNA templates for interrogation by an optical interrogation apparatus, such as a fluorescence detection apparatus, wherein the optical interrogation apparatus can be moveable relative to the PCR station assembly to individually read fluorescence emissions from particular detection windows aligned with the multiple sites.
  • an optical interrogation apparatus such as a fluorescence detection apparatus
  • the multi-site sample holder 100 comprises a body 102 having a top surface 104 and a bottom surface 106.
  • the bottom surface 106 may be a planar surface.
  • the body 102 may be manufactured from an optically transparent or translucent material such as glass or plastic. Molded plastics may include transparent (e.g., thermoplastic materials) or translucent materials (e.g., white or frosted) plastics that are compatible with the particular PGR master mix, primer, and/or reagents being used.
  • Example materials may include elastomers and olefins, polyvinyl chloride (PVC), polycarbonate, polyethylene terephthalate glycol (PETG), styrene, polyethylene, polystyrene, acrylonitrile butadiene styrene (ABS), and polypropylene or combinations.
  • PVC polyvinyl chloride
  • PETG polyethylene terephthalate glycol
  • styrene polyethylene
  • polystyrene acrylonitrile butadiene styrene
  • ABS acrylonitrile butadiene styrene
  • Polypropylene or combinations Polycarbonate and polypropylene are excellent choices for inert and transparent/translucent properties for PCR processing.
  • White and frosted materials may be used for qPCR.
  • the body 102 can be injection molded, compression molded, or any other suitable formation method.
  • the body 102 further comprises an inlet groove 108 formed into the bottom surface 106 and an outlet groove 110 also formed into the bottom surface 106.
  • the outlet groove 110 is positioned alongside the inlet groove 108.
  • the groves 108, 110 may run in parallel to one another and may be molded.
  • the inlet groove 108 and outlet groove 110 can be made as small as possible while still providing good flow.
  • the grooves 108, 110 can have groove depths of from 0.4 mm to 0.7 mm, for example.
  • Grooves 108, 110 can have a variable groove width such as from 0.3 mm to 1.5 mm, for example. Other suitable dimensions may be used.
  • the body 102 further comprises a detection recess 112 formed into the bottom surface 106.
  • the detection recess 112 is sufficiently large for the particular optical interrogation system 340 to be able to measure light emission readings (e.g., fluorescence emission readings) therefrom, for example.
  • the detection recess 112 may have a U-shape of substantially constant width W along the length L of the U.
  • the width W of the detection recess 112 may be from about 1.0 mm to 2.5 mm.
  • the depth D of the detection recess 112 may be from about 0.4 mm to 0.7 mm.
  • the length L along the centerline of the detection recess 112 can be from 5.0 mm to 10.0 mm, for example.
  • the number of detection sites may be two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or even none or more.
  • the total detection area in the detection recess 112 may be greater than 5.0 mm 2 , greater than 7.5 mm 2 , greater than 10.0 mm 2 ' greater than 15 mm 2 , or even greater than 20 mm 2 in some embodiments.
  • Several examples having a detection recess 112 of the configuration shown in FIG. ID are shown in Table 1 below.
  • the sample volume of the PCR solution 124 contained in the detection recess 112 can be less than or equal to 20m ⁇ or from 3m1 to 20m ⁇ , for example.
  • the inlet groove 108 leads to and is connected to the detection recess 112 and the outlet groove 110 is connected to and leads away from the detection recess 112.
  • the inlet and outlet groves 108, 110, and the detection recess 112 may be of the same depth from the bottom surface 106.
  • a fill port 114 may be interconnected to at least the inlet groove 108 (or both the inlet groove 108 and the outlet groove 110) and can provide both a fill and overflow function.
  • the top surface 114S of the fill port 114 may be a planar surface and can be located above the top surface 104 and thus can be sealed with any suitable sealing membrane once the DNA solution 124 is received therein. Sealing may occur prior to insertion of the multi-site sample holder 100 in the PCR station assembly 225 or after insertion therein. Sealing may be by any suitable method.
  • the multi-site sample holder 100 can include a cover 116 connected to and sealed to the bottom surface 106 of the body 102 by any suitable means.
  • the cover 116 can be a thin film cover in some embodiments, such as a generally planar sheet of constant thickness plastic or metal film. The thickness may be between 0.05 mm and 0.5 mm, for example. Other suitable thicknesses and other non-planar configurations of the cover 116 may be used.
  • the cover 116 may be translucent or transparent if the light detector is positioned below the body 102 and cover 116, or opaque if the light detector 350 is positioned above the body 102.
  • the cover 116 can be opaque, such as a black plastic.
  • the cover 116 interfaces with the body 102 to close the bottoms of the inlet groove 108, detection recess 112, and outlet groove 110 and thus form interconnected inlet channel 118, reservoir 119 containing multiple sites (e.g., sites 120A-120E), and outlet channel 122 (see FIGs. IF and 1G).
  • the multi-site feature allows for detection to be carried out at multiple detection sites 120.
  • Each of the sites e.g., sites 120A-120E
  • the master mix can be provided at suitable locations within the sites 120, such as provided on the surface of the cover 116 at locations of the multiple sites 120, or on the top of the reservoir 119, or both.
  • the desired chemistry may be provided by a dry down method, i.e., by applying the chemistry as a spray or gel and then drying the chemistry sufficiently to retain it in place.
  • Barriers or dams or other retaining structures may be formed around or between the respective sites 120A-120E so as to still allow flow of the PCR solution 124 thereto and thereby, but to help prevent movement of the applied desired chemistry to other adjacent downstream sites 120.
  • the sites 120A- 120E may be provided at the bottom of a surrounding recess or wall that is less deep than the depth of the reservoir 119.
  • the outlet channel 122 is interconnected to the reservoir 119 and the included multiple sites 120 therein and may also be interconnected to the fill port 114 in some embodiments.
  • the cover 116 may be bonded to the body 102 by any suitable means.
  • the cover 116 may be bonded by thermal bonding, ultrasonic welding, adhesive bonding, solvent bonding, or by including a pressure sensitive adhesive layer on the body 102. If a metal layer is used, and additional bonding layer of polymer can be used for thermal bonding.
  • sealing of the PCR solution 124 contained in the reservoir 119 can involve at least one sealing member comprising heat sealing or deformation sealing along the lengths of one or more of the inlet channel 118 and outlet channel 122, or providing a sealing member such as a sealing film 333 (FIG. 3) sealing the top of the fill port 114 prior to testing, for example.
  • Other sealing means may be used prior to PCR processing, such as deformation of the ports 123, 126, sealing of the ports 123, 126 by adhesive, filler, or plugs, or sealing with heavy oil (e.g., a mineral oil) in ports 126, 126 or channels 118, 122.
  • the thermal formed seals can be at one or more discreet locations along the lengths of each of the channels 118, 122 at locations that can minimize displacement of the plastic volume and maintain acceptable flatness.
  • the grooves 108, 110 may be locally modified to allow improved sealing.
  • more than one sealed area may be provided along each of the channels 118, 122 to provide for a primary and secondary seal for backup.
  • the seals may avoid trapped air in the channels 118, 122.
  • the second seal can be used to contain any displaced solution 124 that has been displaced by the first seal.
  • the fill port 114 can include one or more funnels (FIGs. IB and 1C) connected to each of the inlet and outlet ports 123, 126.
  • the included cone angle can be less than 120 degrees, for example. The funnels aid in ensuring proper fill with PCR solution 124.
  • the fill port 114 receives the PCR solution 124 via a pipette or other liquid dispensing mechanism into an inlet port 123 of the inlet channel 118.
  • the PCR solution 124 is a suitable solution allowing amplification of the DNA/RNA templates extracted via conventional sample processing operations involving lysis, solid support binding, washing, and elution.
  • the PCR solution 124 can include PCR master mix, DNA/RNA templates, a primer (or probe), possibly a suitable reagent, and possibly water.
  • a PCR master mix is a premixed concentrated solution that has all or some of the components for a real-time PCR reaction that are not sample-specific.
  • a master mix is a solution that can contain a thermostable DNA/RNA polymerase, dNT'Ps, MgCl, and other additives in a buffer optimized for efficient PCR amplification of the DNA/RNA templates.
  • the chemistry provided at the sites may include some of the PCR chemistry.
  • the PCR master mix may include some of the needed chemistry and the sites may include the rest of the needed chemistry.
  • FIG. 1G shows the flow path for the PCR solution 124 in isolation for illustration purposes.
  • the PCR solution 124 flows from the inlet port 123 through the inlet channel 118 and then into and fills the reservoir 119 and the multiple sites 120 (e.g., detection sites 120A-120E).
  • the PCR solution 124 can then exit through the outlet channel 122, which connects to the outlet port 126, which can be, but need not be, located in the fill port 114.
  • Outlet port 126 functions as a vent.
  • the inlet port 123 may be approximately the size of the pipette tip and air 127 may be inserted into the inlet channel 118 to move the PCR solution 124 further into the inlet channel 118, so as to further minimize the amount of the PCR solution 124 needed to be used.
  • the total volume of the inlet channel 118, reservoir 119, and outlet channel 122 together can be less than 30 mR, and from 3 mR to 30 mR in some embodiments, or even from 3 mR to 20 mR in other embodiments.
  • the fill port 114 can include a volume sufficient to reduce any splashing and ensure proper fill. If the inlet channel 118 and outlet channel 122 are sealed by heat sealing, the heat sealing should be close to the liquid-air interface, such as 1 mm to 2 mm therefrom to minimize any trapped air.
  • the inlet channel 118 can comprise an inlet channel first portion 118A and an inlet channel transition portion 118T.
  • the inlet channel transition portion 118T allows transition of the inlet first channel portion 118A smoothly to the larger reservoir 119.
  • the transition portion 118T allows the PCR solution 124 to expand to the larger area reservoir 119 with less turbulence that might undesirably introduce bubbles in the PCR solution 124.
  • the outlet channel 122 can comprises an outlet first channel portion 122A and an outlet channel transition portion 122T.
  • the outlet transition portion 122T allows the transition from the larger reservoir 119 to the outlet first channel portion 122A.
  • the transition portion 122T allows the PCR solution 124 to contract to minimize the amount of PCR solution 124 used therein.
  • the volumes of the inlet channel 118 and outlet channel 122 should be made as small as possible, while not appreciably impeding flow.
  • the multiple sites 120 receive a volume of the PCR solution 124 within the reservoir 119 such that the PCR solution 124 overlies each of the respective multiple sites 120A-120E that are viewable by the optical interrogation apparatus 340, as shown in FIG. 3.
  • FIGs. 2A-2F illustrates a PCR station assembly 200 of a PCR station 225 with an example multi-site sample holder 100 mounted therein.
  • the PCR station 225 is a fixture configured to hold the multi-site sample holder 100 during portions of the PCR processing including the amplification phase and detection phase.
  • Amplification phase involves a large number of heating and cooling steps provided by suitable heating and cooling from a temperature-controlling element 234 (FIGs. 2A, 2F, and 3) wherein DNA/RNA templates are replicated.
  • Detection involves interrogation with the optical interrogation apparatus 340, such as the interrogation apparatus 340 shown in FIG. 3.
  • a fluorescence detection interrogation apparatus is shown in FIG. 3; however, other suitable configurations and types of optical interrogation apparatus may possibly be used.
  • the PCR station 225 can include a base 230 and a clamp member 232.
  • the base 230 and clamp member 232 cooperate to form a recess that is appropriately sized to receive and retain, via clamping, the multi-site sample holder 100 therein.
  • Any suitable clamp initiator 235 such as a screw or mechanical or an automated electromechanical actuator may be used to initiate clamping.
  • the clamping holds the multi-site sample holder 100 stationary in place within the recess and ensures intimate thermal contact of the bottom surface of the multi-site sample holder 100 with the base 230.
  • the base 230 and the clamp member 232 may be made out of any suitable highly thermally-conductive material, such as aluminum, copper, or the like.
  • the PCR station 225 can further include the previously-mentioned temperature-controlling element 234.
  • Temperature-controlling element 234 can be a thermoelectric element, such as a Peltier device that can rapidly heat and cool the base 230 that is in intimate thermal contact with the multi-site sample holder 100.
  • rapid temperature cycling between heating and cooling can be provided as controlled by drive signals provided from a suitable driver which can be part of a controller 342 (FIG. 3).
  • a suitable driver which can be part of a controller 342 (FIG. 3).
  • temperature cycles between a lower nominal temperature of about 55°C and an upper nominal temperature of about 95°C can be implemented.
  • Other suitable upper and lower nominal temperatures can be used depending upon the particular assay or chemistry used.
  • About as used herein means +/- 20%.
  • the PCR station 225 can also include one or more heat sinks 236 coupled thermally to the base 230 and/or possibly to the temperature-controlling element 234.
  • the one or more heat sinks 236 may be coupled to one or more sides or top of the base 230 and/or to the sides and/or bottom of the temperature-controlling element 234. Any suitable construction of the one or more heat sinks 236 may be used.
  • Heat sinks 236 may be aluminum or copper and can include a plurality of suitably-designed fins.
  • FIGs. 2C-2F illustrates the various components of the PCR station assembly 225 including the clamp member 232, the base 230, and the temperature-controlling element 234 in more detail.
  • the clamp member 232 for the multi-site sample holder 100 includes a plurality of viewing apertures 237 formed there through, which define a plurality of viewing windows for the optical interrogation apparatus 340 (FIG. 3) to perform interrogation through.
  • the viewing apertures 237 may include angled side walls 237S, which may be angled at from 30 degrees to 60 degrees to a central axis of the aperture 237, for example.
  • the viewing aperture 237 may be slightly smaller than the dimensions of the reservoir 119 and is the window through which fluorescent emission readings can be taken from each of the multiple detection sites 120.
  • the clamp member 232 can further include a bore 239 formed therein and can be adapted to receive the clamp initiator 235 (e.g., screw or the like). Other automated clamping may optionally be provided.
  • Spring beams 232B can flex and allow the holding portion 232H of the clamp member 232 located outboard from the beams 232B to secure the multi site sample holder 100 in the pocket 230P (FIG. 2E) of the base 230. This the clamp member 232 acts as a spring clip to provide clamping force and ensure good thermal contact between the multi-site sample holder 100 and the base 230.
  • the pocket 230P may include a stop 230S configured to limit the extent of insertion of the multi-site sample holder 100 therein.
  • the pocket 230P can include lateral sidewalls 230W that aid in positioning the reservoir 119 of the multi-site sample holder 100 and sites 120 relative to the viewing apertures 237.
  • the number of viewing apertures can be the same as the number of multiple detection sites 120 of the multi-site sample holder 100.
  • the clamping member 232 shown in FIGs. 2A, 2C, and 2D are designed for a multi-site sample holder 100 having eight detection sites 120 therein.
  • a clamping member 232 that is designed for a multi-site sample holder 100 shown in FIGs. IF and 1G would have five viewing apertures 237.
  • Each of the viewing apertures 120 may be aligned above the location of a corresponding one of the multiple sites 120.
  • the holding portion 232H can register against lateral sidewalls 230W to constrain rotation and to align the multiple sites 120 with the associated viewing apertures 237.
  • Base 230 further can include extenders 230E that extend to the width and length of the temperature-controlling element 234 to maximize the thermal contact and thus maximize thermal conduction therewith so that heating and cooling cycles can be more rapidly conducted.
  • the base 230 may include a temperature sensor 244 therein in thermal contact with the base 230, such as by being mounted therein or thereon.
  • the temperature sensor 244 can be received in a hole formed in the base 230 proximate to the at least some of the multiple sites 120.
  • the temperature sensor 244 may provide feedback information to estimate the temperature of the PCR solution 124 at the multiple detection sites 120.
  • the temperature sensor 244 may be a thermocouple or a thermistor, for example, and may be used by the controller 342 to maintain the desired upper and lower temperatures of the heating and cooling cycles. Other suitable temperature sensors 244 may be used. In some embodiments more than one temperature sensor 244 may be used.
  • FIG. 2F illustrates the temperature-controlling element 234, such as a Peltier device that can be configured to provide rapid heating and cooling cycles to the base 230 and thus to the PCR solution 124 contained in the reservoir 119 above the multiple sites 120 of the multi-site sample holder 100.
  • a PCR testing system 300 is shown and will be fully described.
  • the PCR testing system 300 includes the PCR assembly 200 made up of the PCR station 225, a multi-site sample holder 100, and the optical interrogation apparatus 340.
  • Optical interrogation apparatus 340 is configured to measure the emissions (e.g., fluorescent emissions) from tagged fluorescence (fluorophores) of the PCR solution 124 at one or more wavelengths.
  • the optical interrogation apparatus 340 is an optical system including light source 344 such as a white light LED that projects light through collimating optics (e.g., a suitable lens), through a suitable color filter 345 that cuts out multiple spectra and allows one spectra to pass (e.g., blue light).
  • this blue light spectrum is reflected off from a suitable dichroic mirror 346 and can be focused by focusing optics (e.g., one or more suitable lenses) onto the PCR solution 124 at the particular detection site (e.g., one or 102A-120E) of the multiple sites 120 within the reservoir 119.
  • the point of focus of the optical interrogation apparatus 340 can be moved from one site (e.g., site 120B) of the multiple sites 120 to the next (e.g., 120E) by any suitable moving mechanism, such as by action of one or more actuators (e.g., actuators 347X, 347Y).
  • Actuators 347X, 347Y can act on and move a base plate 343 or like member that the components of the optical interrogation apparatus 340 are affixed thereto.
  • the movements can be responsive to commands from the controller 342 to move in the X direction and/or Y direction (orthogonal to X and Z directions shown), or combinations thereof.
  • Z axis motion capability may also be provided via a z-axis actuator coupled to the base 343.
  • connections of the actuators e.g., actuators 347X 347Y, etc.
  • the optical interrogation apparatus 340 can be selectively moved between each of the respective sites (e.g., sites 120A-120E) to separately and individually obtain fluorescence emission readings from each of the multiple sites 120.
  • a shield member 349 may be used to minimize stray excitation of other sites 120.
  • the reflected blue light causes the tagged fluorophores responsive to the blue light to fluoresce at the site (e.g., one of 120A-120E) of interest upon which the optical interrogation apparatus 340 is focused.
  • This fluorescent emission is then collimated and passed through the dichroic mirror 346, and further filtered with filter 348 to remove any stray excitation light and then focused onto a light detector 350 with focusing optics (e.g., one or more lenses).
  • the relative intensity of the fluorescent emission measured by the detector 350 can render a relative amount of the tagged DNA sequence that is present at that wavelength.
  • analysis of fluorescent emission responses at other wavelengths of excitation light can be provided by changing out at least the filters 345 and dichroic mirror 346 to allow another wavelength of light to pass and obtain measurements thereof.
  • the method 400 includes, in 402, providing a PCR station (e.g., PCR station 225) comprising a base (e.g., base 230), a clamp member (e.g., clamp member 232), and a temperature-controlling element (e.g., temperature-controlling element 234) thermally coupled to the base.
  • a PCR station e.g., PCR station 225
  • a base e.g., base 230
  • a clamp member e.g., clamp member 232
  • a temperature-controlling element e.g., temperature-controlling element 234
  • the method 400 includes, in 404, securing a multi site sample holder (e.g., 100) between the base and clamp member, the multi-site sample holder including an inlet channel (e.g., inlet channel 118) interconnected to a fill port (e.g., fill port 114), a detection region including multiple sites (e.g., multiple sites 120) interconnected to the inlet channel, and an outlet channel (e.g., outlet channel 122) interconnected to the detection region.
  • Outlet channel e.g., outlet channel 122
  • Securing may be by way of a clamp initiator 235 or other suitable clamping or securement means.
  • the method 400 includes, in 406, inserting a volume of PCR solution (e.g., PCR solution 124) into the fill port thus filling the detection region and the multiple sites (e.g., detection sites 120A-120E). Insertion of the volume may be before or after the insertion of the multi-site sample holder 100 in the PCR station assembly 225. Furthermore, before or thereafter, the channels 118, 122, ports 123, 126, and/or fill port 114 may be sealed as described herein.
  • a volume of PCR solution e.g., PCR solution 124
  • Insertion of the volume may be before or after the insertion of the multi-site sample holder 100 in the PCR station assembly 225.
  • the channels 118, 122, ports 123, 126, and/or fill port 114 may be sealed as described herein.
  • the PCR solution 124 is subjected to heating and cooling by subjecting the base 230 to cycles of heating and cooling via the temperature-controlling element 234 to replicate the tagged DNA/RNA templates and to produce tagged replicated DNA/RNA.
  • DNA/RNA as used herein includes DNA and/or RNA).
  • the tagged replicated DNA/RNA are replicates of the DNA/RNA templates provided from the previously- conducted PCR sample processing to extract the templates from the biological sample.
  • fluorescent emissions are detected and measured from the multiple sites (e.g., sites 120A-120E) with an optical interrogation apparatus (e.g., optical interrogation apparatus 340) by exciting the tagged replicated DNA/RNA with a particular wavelength of light.
  • an optical interrogation apparatus e.g., optical interrogation apparatus 340
  • This can be used to monitor the progress of the PCR process.
  • Other wavelengths of light may be used to excite other fluorescent dyes tagged to the tagged replicated DNA/RNA along with associated changes to at least the filters 345 and dichroic mirror 346 in the optical interrogation apparatus 340 for the other wavelengths measured.
  • the measurements from each of the multiple sites 120 may be accomplished by moving the optical interrogation apparatus 340 to respective locations above the respective viewing apertures 237 associated with each respective ones of the multiple sites 120.
  • FIG. 5 illustrates a top, partially sectioned, plan view of an alternate embodiment of multi-site sample holder 500 with multiple sites 520A-520E according to one or more embodiments of the disclosure.
  • an inlet channel 118 and outlet channel 122 are provided, which may connect to inlet port 123 and outlet port 126.
  • the multiple sites 520A-520E can connect to a plenum 555, which couples to the inlet channel 118.
  • Each of the respective multiple sites 520A-520E connects to an outlet distributor 556, which in turn connects to the outlet channel 122.
  • FIG. 6 illustrates a top, partially sectioned, plan view of an alternate embodiment of multi-site sample holder 600 with a collection of closely-spaced multiple sites 620 numbering from 620i through 620 N (where N is an integer) according to one or more embodiments of the disclosure.
  • an inlet channel 118 and outlet channel 122 are provided, which may connect to inlet port 123 and outlet port 126.
  • the multiple sites 620 can be positioned within a reservoir 619, which couples to the inlet channel 118 and outlet channel 122.
  • Each of the respective multiple sites 620c through 620 N can contain a different assay chemistry, which may therefore allow more than one assay to be conducted by the multi-site sample holder 600.
  • the same chemistry may be provided in clusters in more than one site.
  • FIG. 7 illustrates one suitable construction of representative individual sites 620 7 and 620 I5 of the multiple sites 620 for the embodiment of the multisite sample holder 600 of FIG. 6.
  • Each of the multiple sites 620 may include raised walls 758 which can form a hollow cylinder with a closed bottom, wherein the walls 758 can be formed (e.g., injection molded as part of the cover 716, or formed as a honeycomb and otherwise adhered to the cover.
  • Each of the multiple sites 620 can contain assay chemistry, such as a master mix or components thereof that is applied and dried down.
  • individual site 620 7 may include a first chemistry 760 and 620 I5 may include a second chemistry 621 that can be different than the first assay chemistry.
  • FIG. 8 illustrates a top plan view of an alternate embodiment of multi-site sample holder 800 similar to the embodiment of FIG. 1G including multiple sites 120 numbering from 120A-120E according to one or more embodiments of the disclosure.
  • an inlet channel 118 and outlet channel 122 are provided, which may connect to inlet port 123 and outlet port 126.
  • the multiple sites 120 can be positioned within the reservoir 119, which is coupled to the inlet channel 118 and outlet channel 122.
  • Each of the respective multiple sites 120 can contain a different assay chemistry disposed thereat, which may allow more than one assay to be conducted by the multi-site sample holder 800.
  • the assay chemistry can be applied by any suitable means, such as by being sprayed and dried down. Assay chemistry may be applicable to any suitable assay.
  • the inlet port 123 may optionally include a funnel entry 872 and the outlet port 126 may be simply a vent hole alongside the inlet port 123.
  • a robotic grasping feature 874 which can comprise a plurality of notches, formed on either side of the body 102, for example.
  • the notches can have any suitable shape that can be grasped by a robotic gripper, such as the triangular notches shown. Other notch shapes may be used.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Hematology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un porte-échantillon multi-site pour le traitement par PCR. Selon un aspect de l'invention, le porte-échantillon multi-site comprend un corps ayant une entrée et des rainures de sortie formées les unes à côté des autres, un évidement de détection, et un couvercle coopérant avec le corps pour former un canal d'entrée, une région de détection interconnectée au canal d'entrée, et un canal de sortie interconnecté à la région de détection. La région de détection comprend de multiples sites et est configurée pour recevoir une solution de PCR et la réplication se produit dans la région de détection par l'intermédiaire de cycles de chauffage et de refroidissement. Ensuite, les émissions fluorescentes provenant de l'ADN (ou de l'ARN) répliqué marqué dans les multiples sites peuvent être individuellement (ou dans des groupes de moins de tous les sites) détectées et mesurées. L'invention concerne des ensembles de station de PCR, des systèmes de test de PCR et des procédés de fonctionnement d'un système d'essai de PCR, ainsi que d'autres aspects.
PCT/US2021/070133 2020-03-16 2021-02-08 Supports d'échantillons multi-sites, ensembles de station de pcr et procédés de fonctionnement de systèmes d'essai de pcr WO2021189065A1 (fr)

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US202062989952P 2020-03-16 2020-03-16
US62/989,952 2020-03-16

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030027352A1 (en) * 2000-02-18 2003-02-06 Aclara Biosciences, Inc. Multiple-site reaction apparatus and method
US20080190220A1 (en) * 2004-12-23 2008-08-14 Oktavia Backes Novel Microfluidic Sample Holder
US20170314704A1 (en) * 2014-11-14 2017-11-02 Enplas Corporation Liquid handling device
US10195610B2 (en) * 2014-03-10 2019-02-05 Click Diagnostics, Inc. Cartridge-based thermocycler

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030027352A1 (en) * 2000-02-18 2003-02-06 Aclara Biosciences, Inc. Multiple-site reaction apparatus and method
US20080190220A1 (en) * 2004-12-23 2008-08-14 Oktavia Backes Novel Microfluidic Sample Holder
US10195610B2 (en) * 2014-03-10 2019-02-05 Click Diagnostics, Inc. Cartridge-based thermocycler
US20170314704A1 (en) * 2014-11-14 2017-11-02 Enplas Corporation Liquid handling device

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