Portable fluorescence detection system and microassay cartridge

Abstract

Disclosed is a compact, microprocessor-controlled instrument for fluorometric assays in liquid samples, the instrument having a floating stage with docking bay for receiving a microfluidic cartridge and a scanning detector head with on-board embedded microprocessor operated under control of a ODAP daemon resident in the detector head for controlling source LEDs, emission signal amplification and filtering in an isolated, low noise, high-gain environment within the detector head. Multiple optical channels may be incorporated in the scanning head. In a preferred configuration, the assay is validated using dual channel optics for monitoring a first fluorophore associated with a target analyte and a second fluorophore associated with a control. Applications include molecular biological assays based on PCR amplification of target nucleic acids and fluorometric assays in general, many of which require temperature control during detection.

What is claimed is: 
     
         1 . A microassay system for detecting an optical signal in a sample, said system comprising a mechanical system enabled for scanning a detector head across the sample, said detector head comprising at least one detection channel, said detection channel comprising an objective lens and optoelectronic circuit elements configured to capture and amplify optical signals received by said objective lens, wherein said mechanical system is operated by a controller and circuitry external to said detector head and said optoelectronic elements are under control of an autonomous daemon resident in firmware associated with a microprocessor embedded within said detector head. 
     
     
         2 . The microassay system of  claim 1 , wherein said optoelectronic circuit elements are configured for operation in electronic isolation from said external circuitry. 
     
     
         3 . The microassay system of any one of  claim 1  or  2 , wherein said optoelectronic elements are configured for signal processing or signal digitization under control of said daemon. 
     
     
         4 . The microassay system of any one of  claims 1 - 3 , wherein said detector head comprises at least one digital transmitter enabled to transmit said signal in digital form to said external controller. 
     
     
         5 . The microassay system of any one of  claims 1 - 4 , wherein said detector head is enabled to perform a scan across said sample under control of said external controller, and said daemon in said detector head is configured to autonomously construct a map of any optical signals captured during said scan, said map having datapairs expressing signal intensity as a function of location relative to at least one reference point. 
     
     
         6 . The microassay system of  claim 5 , wherein said detector head is configured to scan a sample well on a defined path across a cartridge body, and said at least one reference point is a spatial coordinate on said defined path associated with a registration feature of said cartridge body. 
     
     
         7 . The microassay system of  claim 6 , wherein said registration feature triggers a mechanical, electrical, or an optical switch to initialize said reference point of said map. 
     
     
         8 . The microassay system of  claim 5 , wherein a location of said sample well is determined as a distance from said reference point along said defined path where a first change in optical signal is detected, said first change in optical signal correlating with a first edge of an empty sample well, and a second change in optical signal is detected, said second change correlating with a second edge of an empty sample well under control of said autonomous daemon. 
     
     
         9 . The microassay system of  claim 8 , wherein said scan across said cartridge body is repeated with a sample in said sample well, and a change in optical signal associated with the map coordinates between said first edge and said second edge is analyzed for a change in signal indicative of an assay result under control of said autonomous daemon. 
     
     
         10 . The microassay system of  claim 9 , wherein said change in signal is subjected to electronic conditioning and amplification prior to digitization to produce a digitized signal under control of said autonomous daemon. 
     
     
         11 . The microassay system of  claim 9 , wherein said change in signal is subjected to threshold subtraction prior to digitization. 
     
     
         12 . The microassay system of  claim 9 , wherein said change in signal is subjected to single-bit digitization. 
     
     
         13 . The microassay system of  claim 10 , wherein said digitized signal is accumulated in a register as a series of datapairs during said scan across said well and a summation of signals for said scan is reported to said external controller. 
     
     
         14 . The microassay system of  claim 10 , wherein said digital signal is reported to said external controller. 
     
     
         15 . The microassay system of  claim 5 , wherein said detector head is configured to scan a sample well on a defined path across a cartridge body, and said at least one reference point is an spatial coordinate on said defined path associated with a registration feature of said sample well. 
     
     
         16 . The microassay system of  claim 15 , wherein the daemon is configured to acquire said reference point on said map by detecting a change in optical signal associated with a first edge of a sample well, and the location of said sample well is determined as a distance from said reference point along said defined path to a point at which a second change in optical signal is detected, said second change correlating with a second edge of an empty sample well. 
     
     
         17 . The microassay system of  claim 8 , wherein said scan across said cartridge body is repeated with a sample in said sample well, and a change in optical signal associated with the map coordinates between said first edge and said second edge is analyzed for a change in signal indicative of an assay result under control of said autonomous daemon. 
     
     
         18 . The microassay system of  claim 17 , wherein said change in signal is subjected to electronic conditioning and amplification prior to digitization to produce a digitized signal under control of said autonomous daemon. 
     
     
         19 . The microassay system of  claim 17 , wherein said change in signal is subjected to threshold subtraction prior to digitization. 
     
     
         20 . The microassay system of  claim 17 , wherein said change in signal is subjected to single-bit digitization. 
     
     
         21 . The microassay system of  claim 18 , wherein said digitized signal is accumulated in a register as a series of datapairs during said scan across said well and a summation of signals for said scan is reported to said external controller. 
     
     
         22 . The microassay system of  claim 18 , wherein said digital signal is reported to said external controller. 
     
     
         23 . The microassay system of  claim 15 , wherein said registration feature is an edge having autofluorescence, said edge bordering said sample well. 
     
     
         24 . The microassay system of any one of  claims 1 - 23 , wherein said optical signal is a fluorescent signal and said detector channel comprises an LED for irradiating said mixture with an excitation light, a lightpath having an excitation filter, emission filter, dichroic mirror tuned to enable the detection of emissions from a fluorophore in a defined passband, said objective lens for condensing said excitation light and for collecting said emissions, a sensor with sensor lens for receiving said passband emissions, and a shielded preamplifier and multistage amplifier circuit for amplifying an output from said sensor. 
     
     
         25 . The microassay system of  claim 24 , wherein said amplifier is a tri-stage amplifier and said amplifier is shielded from electronic noise by a Faraday cage, a bypass capacitor, a signal conditioning pre-amplifier, a separate ground plane, a metallized detector head housing, or a combination thereof, and said amplifier is in electrical proximity to said sensor. 
     
     
         26 . The microassay system of  claim 25 , wherein said tri-stage amplifier has a gain of up to 10 14 . 
     
     
         27 . The microassay system of  claim 25 , wherein said tri-stage amplifier has a gain of 10 12  to 10 14 . 
     
     
         28 . The microassay system of  claim 25 , wherein said objective lens is configured for focusing said excitation light on a liquid sample having a mixture of fluorophores and said excitation light is focused on a spot smaller than said sample well. 
     
     
         29 . The microassay system of  claim 28 , wherein said detector head comprises a plurality of detector channels, each said detector channel comprises an source LED for irradiating said sample well with an excitation light, a lightpath having an excitation filter, emission filter, dichroic mirror tuned to enable the detection of emissions from a fluorophore in a defined passband, said objective lens for condensing said excitation light and for collecting said emissions, a sensor with sensor lens for receiving said passband emissions, a shielded preamplifier and multistage amplifier circuit for amplifying an output from said sensor; and said daemon is configured for strobing each said source LED at a frequency so that no two source LEDs are simultaneously on, and for multiplexedly analyzing the signal from each sensor of said plurality of detector channels according to whether the corresponding LED is on or off. 
     
     
         30 . The microassay system of  claim 29 , wherein each said LED is turned on and off at a frequency of 130 Hz. 
     
     
         31 . The microassay system of  claim 30 , wherein said objective lens is configured for focusing an excitation light on a liquid sample containing a mixture of fluorophores. 
     
     
         32 . The microassay system of  claim 29 , wherein said daemon is configured to tabulate a digitized amplifier output and a scanning position in volatile memory within said detector head. 
     
     
         33 . The microassay system of  claim 29 , wherein said daemon comprises instructions for comparing a baseline scan of said sample well with a scan of said sample well containing the mixture, and reporting a difference indicative of a detection event associated with detection of at least one fluorophore-specific emission. 
     
     
         34 . The microassay system of  claim 33 , wherein said daemon is enabled to digitally report said detection event to said host controller. 
     
     
         35 . The microassay system of  claim 29 , wherein said system comprises a graphical user interface and programmable instructions in non-volatile memory for displaying said detection event under control of said external controller. 
     
     
         36 . The microassay system of  claim 29 , wherein said system comprises an I/O port and programmable instructions in non-volatile memory for communicating said detection event to a remote device under control of said external controller. 
     
     
         37 . The microassay system of  claim 29 , wherein said mixture of fluorophores comprises a first fluorophore for detecting a target analyte and a second fluorophore for detecting a control analyte, and further wherein said target analyte and said control analyte are present together in the mixture. 
     
     
         38 . The microassay system of any one of  claims 1 - 37 , wherein said sensor is a photodiode. 
     
     
         39 . The microassay system of  claim 24 , wherein said irradiated light received by said objective lens is formed as a divergent beam, a convergent beam, or a collimated beam for transition therethrough. 
     
     
         40 . A method for automating a microassay, the method comprising operationally dividing the microassay system into fluidic electromechanical and thermal processes controlled by a host controller and optoelectronic processes controlled by an autonomous daemon resident in a scanning detection head, wherein the motion of the scanning detection head across a sample is controlled by the host controller and any optical signal acquisition, preconditioning, amplification and digitization of a signal generated in the sample is controlled by the autonomous daemon. 
     
     
         41 . An apparatus for performing a microassay, the apparatus comprising an assay system operationally divided into fluidic electromechanical and thermal processes controlled by a host controller and optoelectronic processes controlled by an autonomous daemon resident in a scanning detection head, wherein the motion of the scanning detection head across a sample is controlled by the host controller and any optical signal acquisition, preconditioning, amplification and digitization of one or more signals collected from the sample during a scan is controlled by the autonomous daemon. 
     
     
         42 . The apparatus of  claim 41 , wherein said daemon is enabled to multiplexedly collect said one or more signals through a plurality of optical channels. 
     
     
         43 . The apparatus of  claim 42 , wherein said daemon is enabled to condition and digitize said signals during said scan, recording a single bit datum as a function of position or time. 
     
     
         44 . The apparatus of  claim 43 , wherein said daemon is enabled to condition and statistically evaluate digitized optical signals from said plurality of optical channels and to report a detection event after validation of the scan by detection of a control signal in the sample. 
     
     
         45 . The apparatus of  claim 44 , wherein said daemon is enabled to report said detection event to an external device. 
     
     
         46 . The apparatus of  claim 45 , wherein said plurality of optical channels is configured for detecting a plurality of fluorophores comprising a first fluorophore for detecting a target analyte and a second fluorophore for detecting a control analyte, and further wherein said target analyte and said control analyte are present together in the sample. 
     
     
         47 . A microassay cartridge for performing a sample assay, said cartridge comprising
 a) a first circuit card having a hydraulic circuit enclosed therein, wherein said hydraulic circuit of said first circuit card is configured to perform a first sample processing step;   b) an second circuit card having a hydraulic circuit enclosed therein, wherein said hydraulic circuit of said first circuit card is configured to form a second sample analysis step;   c) at least one fluid junction between said hydraulic circuit in said first circuit card and said hydraulic circuit in said second circuit card;   d) an array of pneumatic ports defining a pneumatic interface, each port for receiving a pneumatic pulse applied thereto, at least one said port having a via fluidly joining a pneumatic circuit in said second circuit card to a pneumatic circuit in said first circuit card, said pneumatic circuit in said first circuit card for operating the hydraulic circuit therein, said pneumatic circuit in said second card for operating the hydraulic circuit therein; and,   wherein said cartridge is enabled such that a pneumatic pulse applied to said pneumatic interface at said first via forces said processed sample through said fluid junction from said outboard circuit card to said inboard circuit card.   
     
     
         48 . The microassay cartridge of  claim 47 , wherein said array comprises a plurality of pneumatic ports fluidly connected by vias fluidly joining a pneumatic circuit in said first circuit card to a pneumatic circuit in said second circuit card, such that said plurality of pneumatic ports are enabled to receive a plurality of pneumatic pulses applied according to programmable instructions and to multiplex those pneumatic pulses to fluidic circuit elements of said first circuit card and said second circuit card, thereby reducing the number of ports required to operate said first and said second hydraulic circuits. 
     
     
         49 . The microassay cartridge of  claim 48 , wherein said pneumatic interface is configured to enable multiplexed distribution of stronger pneumatic pulses to circuit elements operative with a stronger pneumatic pressure and weaker pneumatic pulses to circuit elements operative with a weaker pneumatic pressure in said first circuit card and said second circuit card. 
     
     
         50 . The microassay cartridge of  claim 48 , wherein said pneumatic interface is configured to enable multiplexed distribution of positive pressure pneumatic pulses to circuit elements operative with a positive pneumatic pressure and vacuum pressure pneumatic pulses to circuit elements operative with a vacuum pneumatic pressure. 
     
     
         51 . The microassay cartridge of any one of  claims 47 - 50  which comprises a single-use sealing gasket configured to join said pneumatic interface to a host instrument. 
     
     
         52 . The microfluidic cartridge of any one of  claims 47 - 51 , wherein said cartridge is configured for being controlled by applying pneumatic pressure states to the pneumatic circuits, wherein said pneumatic pressure states are selected from a range of positive and negative pressures. 
     
     
         53 . The microfluidic cartridge of  claim 52 , wherein said pneumatic pressure states are selected from about −5 psi, +10 psi, +12 psi, +20 psi, +7 psi, and 0 psi. 
     
     
         54 . The microfluidic cartridge of  claim 52 , wherein at least one pneumatic circuit of said first card and at least one pneumatic circuit of said second card are connected to a common port of said pneumatic interface port array, and said at least one pneumatic circuit in said first card is operative on said sample when said first card receives said sample and said at least one pneumatic circuit of said second card is operative on said sample after said sample is transferred from said first card to said second card. 
     
     
         55 . The microfluidic cartridge of  claim 54 , wherein said first card is configured to extract a nucleic acid extract from a sample, and said second card is configured to analyze said nucleic acid extract for a target analyte. 
     
     
         56 . A microassay apparatus for performing a sample assay, said apparatus comprising
 a) a disposable microassay cartridge configured for docking with a host instrument, said cartridge having at least one hydraulic circuit disposed therein, wherein said at least one hydraulic circuit is configured to operate under control of at least one pneumatic circuit interfaced thereto;   b) an array of one or more pneumatic ports defining a pneumatic interface port array, wherein each port is enabled to convey a pneumatic pressure state from said host instrument to a pneumatic circuit;   c) at least one manifold disposed in said host instrument, said manifold having a plurality of pneumatic pressure sources fluidly coupled thereto, wherein a first pressure source is operated to define a first pressure state in said manifold at a first time interval in an assay sequence and a second pressure source is operated to define a second pressure state in said manifold at a second time interval in said assay sequence, and wherein said manifold is fluidly connected to said pneumatic circuit through a port of said pneumatic interface port array; and,   d) instructions for pneumatically controlling said at least one hydraulic circuit, wherein said instructions are executed under control of a microprocessor having one or more memory elements, and said instructions comprise steps for actuating said first pressure source and said second pressure source according to said assay sequence.   
     
     
         57 . The microassay apparatus of  claim 56 , wherein said cartridge comprises at least one hydraulic circuit configured to operate under control of a plurality of pneumatic circuits interfaced thereto, said pneumatic interface port array having fluidic connections to said at least one manifold, wherein said at least one manifold is configured to be operated at a plurality of pressure states. 
     
     
         58 . The microassay apparatus of  claim 57 , wherein said at least one hydraulic circuit is configured to be operated under pneumatic control of pressure states conveyed by a plurality of pneumatic circuits, at least one of said pneumatic circuits having a plurality of pressure states. 
     
     
         59 . The microassay apparatus of any one of  claims 56 - 58 , which comprises a single-use sealing gasket configured to join said pneumatic interface port array of said cartridge to said host instrument. 
     
     
         60 . A host instrument for removably receiving a microassay cartridge, said instrument comprising:
 a) a supporting baseplate;   b) a docking bay for receiving a microassay cartridge, wherein said docking bay is mounted on said baseplate;   c) a pneumatic control manifold disposed within said baseplate, said manifold having disposed thereon a pneumatic interface port array within said docking bay;   d) attached to said baseplate within said docking bay, a heater assembly comprising at least one heating member with spring mount and with superior surface dimensioned for interfacing with an undersurface of said cartridge; and,   wherein said docking bay comprises a clamping mechanism configured to reposition the cartridge within the docking bay from first position above the pneumatic interface port array to a second position wherein said cartridge is operatively engaged with said pneumatic interface port array, and further wherein said spring mount is configured to press an undersurface of the microfluidic cartridge in said second position against said superior surface of said heating member.   
     
     
         61 . The apparatus of  claim 60 , wherein said spring mount exerts a spring force of about 1 psi over a heat transfer surface reversibly contacting said cartridge and said heating member. 
     
     
         62 . The host instrument of any one of  claims 60 - 61 , wherein said docking bay with clamping mechanism is configured to simultaneously engage the pneumatic interface port array of the baseplate with mating ports of the microassay cartridge and to press the cartridge against the at least one spring-mounted heating member. 
     
     
         63 . The host instrument of any one of  claims 60 - 62 , wherein said host instrument is configured for pneumatically controlling the operations of the microassay cartridge via said pneumatic control manifold while clamped in said docking bay against the heating member. 
     
     
         64 . The host instrument of any one of  claims 60 - 63 , wherein said clamping mechanism is configured for disengaging said cartridge from said pneumatic interface port array and said heating mechanism of said baseplate so that said cartridge may be removed from said docking bay. 
     
     
         65 . The host instrument of  claim 64 , wherein said cartridge comprises a single-use gasket configured to sealingly join said cartridge to said pneumatic interface port array of said docking bay. 
     
     
         66 . The host instrument of any one of  claims 60 - 65 , wherein said heating member is electropolished and chrome plated. 
     
     
         67 . The host instrument of any one of  claims 60 - 66 , wherein said heating member is resistively heated. 
     
     
         68 . The host instrument of any one of  claims 60 - 66 , further comprising a first fan for convectively dissipating heat from a cooling member disposed on said heating member. 
     
     
         69 . The host instrument of any one of  claims 60 - 67 , further comprising a second fan for convectively cooling a sample well disposed in said microassay cartridge. 
     
     
         70 . The host instrument of  claim 69 , wherein said second fan is configured for performing a thermal annealing function by cooling said sample well while the sample is fluorescently monitored. 
     
     
         71 . A method for co-assaying a sample for a target fluorescent signal associated with a pathogenic condition and a control fluorescent signal associated with an endogenous non-pathogenic component of said sample, said method comprising:
 a) scanning a sample well with a detector head of  claim 1 , wherein said target fluorescent signal, if present, is detected in a first optical channel of said detector head and said control fluorescent signal, if present, is detected in a second optical channel of said detector head; and   b) reporting a valid result of said assay if and only if said second fluorescent signal is detected.   
     
     
         72 . A method for performing a FRET melt curve determination, comprising
 a) heating a sample chamber containing a FRET probe and a target nucleic acid sequence to a temperature above the melt temperature of the duplex probe;   b) turning off said heating; and,   c) using a detector head of  claim 1 , monitoring a change in fluorescence of said FRET probe in response to a change in temperature of said sample chamber caused by turning on a fan and directing a stream of ambient air onto the sample chamber, thereby rapidly cooling said sample chamber while monitoring fluorescence.   
     
     
         73 . The method of  claim 72 , wherein said melting curve is completed in less than a minute.