Field portable, handheld, recirculating surface acoustic wave and method for operating the same

Abstract

A system and method for performing a portable, fast, field assay of a small sample biological analyte includes a microfluidic cartridge and a reader with which the microfluidic cartridge is selectively communicated. A closed microfluidic circuit mixes and recirculates the analyte with a buffer. A shear horizontal surface acoustic wave (SAW) detector communicates with the microfluidic circuit and has a plurality of channels including at least one functionalized sensing channel in which the mixed analyte and buffer is recirculated and sensed. Capture of the analyte is amplified by recirculation of the analyte and buffer, and detection is amplified by use of an all-purpose endospore display mass amplification.

Claims

1. A system for detecting an analyte in a small volume biological sample, the system comprising: a shear horizontal surface acoustic wave (SAW) detector with a sensing lane functionalized with an antibody; CED means for convection enhanced delivery (CED) of the sample to the SAW detector, the CED means comprising a distributing manifold and an active mixer coupled to the distributing manifold for uniformly distributing the mixture to increase kinetics of the analyte within a buffer solution for actively recirculating the biological sample through the system for a predetermined number of cycles in a predetermined time period to reduce time required for detection by increasing the probability of encounter of the analyte with the antibody in the sensing lane of a SAW detector; a SAW interface circuit; and a microcontroller, where the SAW interface circuit comprises a clock oscillator, an RF synthesizer coupled to the clock oscillator, a low pass filter and splitter having an input coupled to the RF synthesizer and an output coupled to the SAW detector, a phase/gain detector coupled to the low pass filter and the splitter and having a data input coupled to the SAW detector, an analog-to-digital converter having an input coupled to an output of the phase/gain detector and having an output coupled to the microcontroller, a pump driver, and a motor with a motor driver for mechanically loading a microfluidic cartridge.

2. The system according to claim 1, wherein the distributing manifold comprises: a plurality of channels associated with the SAW detector; and a closed microfluidic circuit in communication with the SAW detector, wherein the closed microfluidic circuit further comprises the active mixer coupled to a microfluidic reservoir chamber for mixing the analyte and buffer into a homogeneous mixture.

3. The system of claims 2, further comprising a pump chamber into which the analyte is transmitted from the microfluidic reservoir chamber.

4. The system of claim 2, wherein the closed microfluidic circuit comprises in sequence: the microfluidic reservoir chamber for mixing the analyte and the buffer; means for delivering a biological mass amplifier to modify the mass of the analyte detectable in a sensing channel to meet a limit of detection (LOD) of the SAW detector with a predetermined size of the sample; and/or means for delivering a detergent solution to retain quantitative capability of biosensor measurement and minimize non-specific binding.

5. The system of claim 1, where the distributing manifold and active mixer coupled to the distributing manifold for actively recirculating the biological sample through the system for a predetermined number of cycles in a predetermined time period to reduce time required for detection by increasing the probability of encounter of the analyte with the antibody in the sensing lane of a SAW detector is configured for detecting the analyte in the small volume sample of biological sample, when the small volume sample comprises 100 or less molecules of the analyte.

6. The system of claim 1, where the distributing manifold and active mixer coupled to the distributing manifold for actively recirculating the biological sample through the system for a predetermined number of cycles in a predetermined time period to reduce time required for detection by increasing the probability of encounter of the analyte with the antibody in the sensing lane of a SAW detector is configured for detecting the analyte in the small volume sample of biological sample, when the small volume sample comprises 10 or less molecules of the analyte.

7. The system of claim 1, where the distributing manifold and active mixer coupled to the distributing manifold for actively recirculating the biological sample through the system for a predetermined number of cycles in a predetermined time period to reduce time required for detection by increasing the probability of encounter of the analyte with the antibody in the sensing lane of a SAW detector is configured for detecting the analyte in the small volume sample of biological sample, when the small volume sample comprises one molecule of the analyte.

8. The system of claim 1 where the distributing manifold and active mixer coupled to the distributing manifold for actively recirculating the biological sample through the system for a predetermined number of cycles in a predetermined time period to reduce time required for detection by increasing the probability of encounter of the analyte with the antibody in the sensing lane of a SAW detector render the limit of detection (LOD) of the SAW detector at 1 picogram of analyte.

9. The system of claims 1, where the biological sample comprises a virus, Ab-Conjugated or scFv-conjugated endospore specifically attached to the analyte through an ELISA chain.

10. The system of claim 2, where the closed microfluidic circuit recirculates the mixed analyte and buffer through the closed microfluidic circuit a multiplicity of times within a predetermined time period less than or equal to one hour in duration.

11. The system of claim 2, where the closed microfluidic circuit recirculates the mixed analyte and buffer through the closed microfluidic circuit approximately 60 times within an approximately 5 minute duration.

12. The system of claim 4, where the analyte and the buffer are combined in the microfluidic reservoir chamber using a sample size of the order of 50 μL in an amount of buffer of the order of 100 μL.

13. The system of claim 1 further comprising a processor for processing a signal from the analyte, where the processor is comprised of a reader, and where the SAW interface circuit, the microcontroller for controlling the SAW detector through the SAW interface circuit, and a user interface associated with the microcontroller are disposed in the reader.

14. A system for detecting an analyte in a small volume biological sample, the system comprising: a shear horizontal surface acoustic wave (SAW) detector with a sensing lane functionalized with an antibody; and CED means for convection enhanced delivery (CED) of the sample to the SAW detector, the CED means comprising a distributing manifold and an active mixer coupled to the distributing manifold upstream from the SAW detector for uniformly distributing the mixture to increase kinetics of the analyte within a buffer solution in the SAW detector and for-actively recirculating the biological sample through the SAW detector.

15. The system according to claim 14, wherein the distributing manifold comprises: a plurality of channels associated with the SAW detector; and a closed microfluidic circuit in communication with the SAW detector, wherein the closed microfluidic circuit further comprises the active mixer coupled to a microfluidic reservoir chamber for mixing the analyte and buffer into a homogeneous mixture.

16. The system of claim 14, where the distributing manifold and active mixer coupled to the distributing manifold actively recirculates the biological sample through the system for a predetermined number of cycles in a predetermined time period to reduce time required for detection by increasing the probability of encounter of the analyte with the antibody in the sensing lane of a SAW detector renders the limit of detection (LOD) of the SAW detector at 1 picogram of analyte, or is configured for detecting the analyte in the small volume sample of biological sample, when the small volume sample consists of a sample size selected from one molecule, 10 or less molecules, or 100 or less molecules of the analyte.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a flow diagram of the functional operations of the microfluidic cartridge and the reader of the current invention.

(2) FIG. 2 is a perspective top partial cross-sectional view of the microfluidic cartridge wherein its internal components are shown.

(3) FIG. 3 is a flow chart of the operation of the microfluidic cartridge of the current invention illustrating the recirculation cycle.

(4) FIG. 4 is a block diagram of the electronic components of the microfluidic cartridge and reader of the current invention.

(5) FIG. 5 is a longitudinal side cross-sectional view of the microfluidic cartridge seen in FIG. 2 illustrating the three levels of structure of the cartridge.

(6) FIG. 6 is a simplified top perspective partial cross-sectional view of the microfluidic cartridge seen in FIG. 5 illustrating the combination of components employed in the recirculation protocol.

(7) FIG. 7A is a top perspective view of the microfluidic cartridge seen in FIG. 2.

(8) FIG. 7B a bottom perspective view of the microfluidic cartridge seen in FIG. 7A.

(9) FIG. 7C a top plan view of the microfluidic cartridge seen in FIG. 7A.

(10) FIG. 7D a side planar view of the microfluidic cartridge seen in FIG. 7A.

(11) FIG. 8 is a partial side cross-sectional view of microfluidic cartridge showing the relationship of the manifolds, the SAW detector, and the printed circuit board used within the microfluidic cartridge.

(12) FIG. 9 is a top down perspective view of the internal components of the portable handheld field assay of the current invention.

(13) FIG. 10 is a bottom up perspective view of the portable handheld field assay seen in FIG. 9.

(14) FIG. 11A is a top perspective view of the cartridge loader/carrier and its mechanical components.

(15) FIG. 11B is a side plan view of the cartridge loader/carrier and its mechanical components seen in FIG. 11A.

(16) FIG. 12 is an exploded perspective view of the microfluidic cartridge showing seen in FIG. 2.

(17) FIG. 13A is an illustration of a comparison of whole IgG with F(ab′)2 and scFv fragments used for biological mass amplification for SH-SAW biosensor technology.

(18) FIG. 13B is an illustration of common linker proteins used to bind IgG and scFv fragments used for biological mass amplification for SH-SAW biosensor technology.

(19) FIG. 13C is an illustration of an endospore display system used to bind any IgG or scFv for mass amplification.

(20) FIG. 14A is a flow diagram illustrating the delivery of an analyte using recirculation within a multi-reservoir system and a magnified view of the analyte interacting with an antibody disposed on the SAW of the current invention.

(21) FIG. 14B is a flow diagram illustrating the delivery of a biological mass amplifier using recirculation within a multi-reservoir system and a magnified view of the biological mass amplifier interacting with the analyte.

(22) FIG. 14C is a flow diagram illustrating the delivery of dilute detergent to wash off non-specifically bound entities using recirculation within a multi-reservoir system and a magnified view of excess biological mass amplifier being removed from the SAW.

(23) FIG. 15A is an illustration of protein-G orientation-enabled detection of engineered PhiX174-HA virus using the microfluidic cartridge of the current invention.

(24) FIG. 15B is an illustration of detection of all-purpose endospore resulting in mass amplification using the microfluidic cartridge of the current invention.

(25) FIG. 15C is an illustration of a simplified strategy for detection of PhiX174-HA virus for initial studies using the microfluidic cartridge of the current invention.

(26) FIG. 15D is an illustration of a simplified strategy for detection and proof-of-concept of endospore-enabled mass amplification for initial studies using the microfluidic cartridge of the current invention.

(27) The disclosure and its various embodiments can now be better understood by turning to the following detailed description of the preferred embodiments which are presented as illustrated examples of the embodiments defined in the claims. It is expressly understood that the embodiments as defined by the claims may be broader than the illustrated embodiments described below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(28) FIG. 1 is a system top level block diagram of microfluidic system 10. Microfluidic system 10 includes a disposable microfluidic cartridge 11 which is inserted into and read by a reader 13. The microfluidic cartridge 11 in turn includes a shear wave surface acoustical wave detector (SAW) 12 and a temperature sensor, micropump and mixer assembly 15. Various embodiments of SAW detector 12 are described in such as described in PCT Patent Application serial no. PCT/US17/48055, entitled Surface Acoustic Wave Biosensor Employing An Analog Front End And DNA Encoded Libraries To Improved Limit Of Detection (LOD) With Exemplary Apparatus Of The Same, filed on 22 Aug. 2017, incorporated herein by reference in its entirety. Reader 13 includes a signal generator 46 that is coupled to and drives SAW 12 and a signal acquisition circuit 48 coupled to SAW 12 for receiving the data signals output by SAW 12. The operation of signal generator 46 and signal acquisition circuit 48 are coupled to microcontroller 54, which provides signal and data processing control subject to software control. Drivers 52 are also coupled to microcontroller 54 and provide the driving and control signals to the elements of the temperature sensor, micropump and mixer assembly 15. User interface 56 is coupled to microcontroller 54 and includes output displays 58, LEDs 60, switches 62, Wi-Fi/Bluetooth connections 64, and secure digital (SD) card connectors 66 as described below. The circuitry of reader 13 is coupled to and powered by a power or battery source 50.

(29) The elements of disposable microfluidic cartridge 11 denoted in FIG. 1 are illustrated in the diagram of FIG. 2. Disposable microfluidic cartridge 11 includes a reservoir chamber 26 in which the analyte 20 is injected in the field through a Tyvek® membrane 30 using a conventional syringe 31 (Tyvek® is a brand of Dupont for flash spun high-density polyethylene fibers). Reservoir chamber 26 includes buffer 24 and is actively mixed with analyte 20 in reservoir chamber 26 by piezoelectric active mixer 41. The mixed buffer 24 and analyte 20, comprising mixture 28, flow through one-way check valve 68 communicated from reservoir chamber 26 to a pump chamber 70 by active of a piezo-pump 36. Pump chamber 70 has a hydrophobic membrane 40 toward which any air bubbles in the mixture 28 are driven and through which the air bubbles escape to ambient atmosphere. The degassed mixture 28 is then pumped into passive mixer 42 to further even the flow rate and mixing. Mixture 28 is then supplied to manifold through which it is supplied to four parallel channels 74 of SAW detector 12 through nozzle-diffuser combination 32. In the illustrated embodiment each channel 74 is approximately 50 μm high, 1.2 mm wide and 4 mm long. One of the channels 74 is a sensing lane 16, while the remaining three channels 74 are reference lanes 17 of SAW detector 12. Sensing lane 16 and reference lanes 17 are identical with the exception that sensing lane 16 is functionalized with a selected antibody 22 according to the analyte 20 which is being detected. The antibody is preferably mass enhanced by the inclusion of a gold nanoparticle, endospore, magnetic beads, or synthetically coupled mass tags linked thereto. Thus, a fraction of analyte 20 will be captured by the functionalized antibodies 22 in sensing lane 16. Any remaining portion of mixture 28, including all nonhybridized analyte 20 from sensing lane 16 and reference lanes 17, are collected in receiving manifold 33 and recirculated through return line 76 to reservoir chamber 26. In the illustrated embodiment, the pumping rate is selected so that the contents of reservoir chamber 26 is recirculated 12 times each minute. A single sampling or measurement is made in microfluidic cartridge 11 once in five minutes. Thus, during a single measurement cycle, mixture 28 is recirculated through microfluidic cartridge 11 sixty times. Effective amplification of the small sample 18 is therefore solved by repetitive recirculation, mixing and cumulative hybridization of analyte 20. The rate of circulation within the microfluidic circuit can vary based on parameter such as pressure drop, Reynolds numbers, viscosity of the medium, temperature, geometrical terms, and analytes conjugation properties (such as K+, K−), and diffusion terms as generally described by the Navier Stoke equation, coupled with the diffusion term.

(30) The operational phases of microfluidic cartridge 11 can now be better understood by turning to the simplified flow diagram of FIG. 3. Reservoir chamber 26 is loaded in the field with typically 50-100 μL of sample 18 at step 78 from syringe 31 by injection through membrane 30. Active mixer 41 homogenously mixes analyte 20 with buffer 24 in reservoir chamber 26 while the piezo pump 36 positively displaces mixture 28 at step 80 through check valve 68 at step 82. Piezo pump 36 maintains a positive pressure through the entire microfluidic circuit to overcome any pressure drops in the system as denoted at step 84. Mixture 28 enters bubble trap 38 (as seen in FIG. 7) at step 86 and all entrained bubbles are removed from further circulation in the microfluidic circuit. Mixture 28 then flows into passive mixer 42 at step 88, which is a geometric manifold that reduces any remaining inhomogeneity of analyte 20 in buffer 24. Mixture 28 flows into a splitter or nozzle-diffuser combination 32 at step 90 which provides for a balanced distribution of mixture 28 into each of the multiple channels 74 of SAW detector 12. Mixture 28 then flows through sensing lane 16 and reference lanes 17, where the confining 50 μm channel height assures uniform flow across the width of sensing lane 16 at step 92. After exiting channels 74 mixture 28 from each of the channels 74 is combined in receiving manifold 33 and returned under pressure in return line 76 to reservoir chamber 26 at step 94.

(31) FIG. 4 is a simplified block diagram of the circuitry in microfluidic cartridge 11 and reader 13. The circuitry of reader 13 is logically centered around microcontroller 54, which includes related peripherals 95 such as oscillator 96, a real-time clock 98, an on-board temperature sensor 100, memory 102, in-circuit serial programming (ICSP) module 104, and driver 105 coupled to cooling fan 107, all of which are coupled to microcontroller 54.

(32) A power module 106 is also coupled to microcontroller 54 and includes battery source 50, fuel gauge 108 coupled between battery 50 and microcontroller 54, universal serial bus (USB) connector 110 coupled to power management integrated circuit (PMIC) 112 having one output coupled to a low drop out regulator (LDO) 114 and hence all coupled to microcontroller 54. PMIC 112 is also coupled to battery source 50 for charge and voltage monitoring and boost 113 for providing for a boosted DC voltage.

(33) A user interface 116 is coupled to microcontroller 54 and includes in the illustrated embodiment program switches 62, output LEDs 60 with connected driver 61, a six-degrees of freedom inertial measurement unit (IMU) 118 employed by the apparatus to adjust for flow rate relative to gravitational vector in regiments where an orthogonal gravitational vector cannot be achieved is coupled through a serial peripheral interface bus, as is a secure data (SD) card connector 120. A Wi-Fi module 122 is coupled to microcontroller 54 through a universal asynchronous receiver/transmitter (UART) bus, whose output is coupled in turn to an antenna 124 to allow wireless communication by microfluidic system 10 with the internet or other computer network. An oscillator 125, coupled to audio module 126, is coupled to microcontroller 54, whose output in turn is coupled to a speaker 128 so that microfluidic system 10 can communicate with the user through audio messages. Microcontroller 54 is also coupled to a capacitive touch (CAP) display 130 to allow screen touch communication with the user. A thin-film transistor (TFT) color display 132 which is backlight by light 134 is coupled to microcontroller 54 through graphic controller 136, which in turn is supported by an oscillator 140 and synchronous dynamic random access (SDRAM) memory 138 coupled thereto.

(34) As further seen in FIG. 4, a SAW interface 142 is also coupled to microcontroller 54 to provide a control interface between microcontroller 54 and microfluidic cartridge 11. Oscillator 144 provides a clock signal to RF synthesizer 146, whose output is passed through a low pass filter and splitter 148 to drive SAW detector 12. A reference signal is supplied from low pass filter (LPF) and splitter 148 to phase/gain detector 150 coupled to the output of SAW detector 12. The output of phase/gain detector 150 is converted into digital form by analog-to-digital converter (ADC) 152 and provided to microcontroller 54 as the data signal through a serial data bus. Power is provided from PMIC 112 to low noise low drop out regulator (LDO) 154 to RF synthesizer 146 and phase/gain detector 150. The temperature of mixture 28 in microfluidic cartridge 11 is measured by negative temperature coefficient thermistor (NTC) 156 and provided through operational amplifier 158 to analog-to-digital converter (ADC) 160 and hence to microcontroller 54. Photometric measurements are made possible of mixture 28 by means of an RGB LED 162 powered by driver 164 controlled by microcontroller 54. The incoming optical signal is directed to widened optical channel 77 best shown in FIG. 5 in return line 76 by which optical sensing of the recirculating fluid flow can be measured. The returned optical signal is received by photodiode 166, whose output is digitized by ADC 160 and provided to microcontroller 54. The light absorption spectra received from the recirculating analyte is generating a spectral shift proportional to the absorption rate due to protein or any circulating component within the channel. The resulting signal indicates whether the recirculating analyte are passing through the channel while the microcontroller records the optical signal indicating the presence, or lack thereof, of analyte within the channel. One skilled in the relevant art can conceive of an alarm signal and intelligent data gathering associated with such an embodiment as it indicates the presence of suspended analyte concentration. Piezo pump 36 and active mixer 41 in microfluidic cartridge 11 are driven by piezo driver 168 controlled by microcontroller 54. Microfluidic cartridge 11 may also include a biological identification module 170 coupled to microcontroller 54 through ADC 160 by which identification information specific to microfluidic cartridge 11 is read. This safety feature enables a clear distinction by identifying the analyte specificity with a resistor value registered by the resident memory 102 and provides the reader with an analogue distinction of what specific antigen concentration is being recorded. A driver 169 is coupled to microcontroller 54 and thence to a motor 171 for providing for motorized loading of microfluidic cartridge 11 into a cartridge holder 173 for automated and uniform connection of microfluidic cartridge 11 to reader 13.

(35) The arrangement of microfluidic cartridge 11 can be better appreciated by comparing FIGS. 5 and 6, wherein the three levels of the flow path structure of microfluidic cartridge 11 can be visualized. FIG. 5 is a side cross sectional view of microfluidic cartridge 11 and FIG. 6 is a top perspective view of microfluidic circuit in the cartridge 11. The top level includes reservoir chamber 26 and spiral bubble trap 38. Beneath the top level is a middle level which includes passive mixer 42, splitters or manifold 32, receiving manifold 33 and return line 76. Beneath the middle level is the bottom level which includes active mixer 41, check valve 68, pump chamber 70, and channels 74. Thus, it can readily be understood and visualized that the mixture 28 starts in reservoir chamber 26 in the top level and is drawn down into active mixer 41 in the bottom level from where it flows up through check valve 68 in the middle level into pump chamber 70 in the bottom level. Mixture 28 then flows up to the spiral bubble trap 38 in the top level and after being de-bubbled flows back down into the middle level of passive mixer 42. Mixture 28 continues to flow to manifold 32 in the middle level and thence is distributed to channels 74 of SAW detector 12 in the bottom level. From channels 74 mixture 28 is then pumped into receiving manifold 33 and along return line 76 in the middle level to reservoir 26 in the top level.

(36) FIGS. 7A-7D show the externally visible components of the cartridge 11, showing in FIGS. 7A, 7C and 7D the de-bubble chamber 38. FIG. 7A further depicts top RF shield area 198, while FIGS. 7C and 7D show the reservoir 26. FIG. 7B shows gasket gland 183, pump 36 and active mixer 41.

(37) FIG. 8 is a side cross sectional view in enlarged scale of the SAW detector 12 bonded underneath and laser welded to the corresponding adjacent portions of microfluidic manifolds 32 and 33 of microfluidic system 10, which in the illustrated embodiment are made of cyclic olefin copolymer. SAW detector 12 is a conventional LiTaO.sub.3 substrate cut for Love Wave propagation with opposing piezo interdigitated transducers (IDT) 174 on each end of a surface waveguide 176 on which are provided sensing lane 16 and reference lanes 17. The flexible printed circuit board 184 to which SAW detector 12 is adhesively coupled includes a 50 μm Kapton® top layer 202 (Kapton® is a mark of Dupont De Nemours and Co. Corp. of Delaware) underneath which is 35 μm copper cladding 203 followed by a 50 μm Coverlay® bottom layer 204 (Coverlay is a registered mark of Coverlay Mfg Inc. of Texas). Printed circuit board 184 continues from the right end of the partial view of FIG. 8 to include conventional mounting locations for the remaining electrical elements of microfluidic cartridge 11 as described above and for RF ground shielding 198 for SAW detector 12.

(38) FIG. 9 is a perspective view of one embodiment of the field portable reader 13 into which microfluidic cartridge 11 is inserted. In the illustrated embodiment reader 13 is similar in size to a conventional cell phone, namely 25 mm thick, 180 mm long and 100 mm wide. Most of the upper top surface is occupied by the capacitive touch screen 130. Microfluidic cartridge 11 is inserted into a conventional side slot loading receiver (not shown) similar to a slot loading DVD drive in a conventional laptop computer. In this manner, microfluidic cartridge 11 is uniformly handled or loaded into reader 13 and shielded or isolated from the environment without undue force or stress applied thereto and without electrode or contact misalignment errors that might result from manual handling by an untrained user. The user interfaces with the device through a series of buttons 62 that control menu features. For output of data, there exists an SD card reader 120, a micro-usb output port 110 and a speaker 128.

(39) FIG. 10 is a perspective view of microfluidic system 10 from the bottom showing the placement of the battery 50, RF shields 206, active mixer 41 and the fan component 107.

(40) FIG. 11A is a perspective view of the cartridge loader 190 and uninserted cartridge carrier 193. Incorporated into the cartridge loader 190 is a Maxton motor 187 and motor cam 192 for motorized loading and unloading of the cartridges 11. The mechanism is attached by dowel pins 188 to a dowel pin base 189 which binds the apparatus to the PCB assembly 72. FIG. 11B is a side plan view of the cartridge loader 190 with the cartridge carrier 193 inserted therein.

(41) FIG. 12 shows the microfluidic components of the cartridge 11. The reservoir cap 177 fits over the septum 194 to retain sample 18. The de-bubble cap 182 is placed on top of the hydrophobic filter 40 to reduce the number of bubbles that reach SAW 12 surface and effect diffusion timescale. Two piezo seals 178 are incorporated into the active mixer 41 and piezo pump 36 to maintain a closed circuit. SAW 12 is attached to the cartridge 11 by placement on a FPC 196 that holds both the piezo pumps 36, 41 and corresponding chips. Temperature variation is monitored by thermistor 195 reporting to microcontroller 54 as to the actual temperature of the cartridge during the operation and where the microcontroller lookup table residing in memory 102 adjust the flow rate in accordance with Navier Stoke equation. A gasket 181 ensures no sample 18 escapes at the SAW 12 site. A SAW compression bar 179 holds the SAW 12 firmly to the gasket 181, and is held down by two screws 180.

(42) FIG. 13a is a graphical representation of the capture and detection technique employed by the invention whereby a comparison between complete antibodies 300 and scFv fragmented antibodies 301. FIG. 13B indicate how protein A 302 interacts with the complete antibodies 300 and protein L 303 interacts with scFv fragmented antibodies 301. FIG. 13C illustrates how endospores 304 are generated to either express protein A 302 or protein L 303 depending on the capture application, as well as an ELISA chain consisting of an endospore 304, and either protein A 302 and a complete antibody 300 or protein L 303 and a fragmented antibody 301 are conjugated.

(43) FIGS. 13A-13C further illustrate the bio-amplification and mass enhancement endospore 304 of the analyte 20, while illustrating the biochemical sequencing-events performed automatically by the microfluidic chamber 14 and directed by the Reader 13. The strategy to develop all-purpose endospore display system for biological mass amplification for SH-SAW biosensor technology is the purpose of the current invention and it comprises the example shown in FIG. 13A where there is a graphic representation and a comparison of whole IgG antibody with F(ab′)2 300 and scFv fragments 301. FIG. 13B is a graphical representation of a common linker proteins 302 and 303 used to bind IgG and scFv fragments. FIG. 13C demonstrates a bio-amplification technique where an endospore 304 display system is used to bind any IgG 300 or scFv 301 for mass amplification purposes. The technique noted above relates to the fact that concentration of analyte at the range of femtogram to picogram per ml.sup.−1 are below threshold resolution of the SAW 12 and the amplification of mass is a necessary step to obtain a measurement at this range. The ability of the preferred embodiment to measure such concentrations and the use of bio-amplification while obtaining results commensurable with clinical values is main purpose of the current invention.

(44) FIGS. 14A-14C are graphical representations of the preferred embodiment of the SAW 12 whereby a three stage convection enhanced delivery (CED) mechanism that contains the analytes 22 in sample 18 is introduced into chamber 305. In the first step seen in FIG. 14A, the analytes 22 are being recirculated through the reservoir sample complex to the SAW 12 and returned through the return line 76. The next step in FIG. 14B shows the utilization of the reservoir endospore complex 309 to administer the biological mass amplifier Ab-conjugated r scFv conjugated endospores, or any other functionalized mass amplifying particles, from chamber 306 over the SAW 12. The final step in FIG. 14C shows the reservoir detergent complex from chamber 307 being administered to dilute detergent and wash off any non-bound endospores 304 from the SAW 12.

(45) FIG. 15A is a schematic representation and example of a bio-amplification technique used by the current invention demonstrating the ability of mass amplification to employ multiple techniques of adding mass to the SAW 12 employing, for example, viruses such as PhiX174-HA virus 313. Displayed is an initial design of protein-G 311 orientation-enabled detection of an engineered PhiX174-HA virus 313 through an ELISA chain consisting of the silane surface 310, protein G, capture antiHA antibody 312, and the target PhiX174-ha 313. FIG. 15B illustrates the detection using endospore 304 display mass amplification through the creation of an ELISA chain consisting of a silane 310 surface, capture antibody 300, analyte 20, and the Ab-Conjugated or scFv-conjugated endospore 309. FIG. 15C shows a simplified strategy for detection of PhiX174-HA virus 313 for initial studies consisting of a silane surface 310, and capture antiHA antibody 312 and the target PhiX174-HA virus 313. FIG. 15D shows a simplified strategy for detection and proof-of-concept of endospore 304 enabled mass amplification for initial studies consisting of an ELISA chain of silane 310, COTB capture antibody 314, and CotB amplified endospore 315. It is understood that further mass amplification sandwich techniques such as using functionalized magnetic beads, synthetically generated mass tags, gold nano particles, viruses, microphages or bacteriophages, or any other suitable mass employing an ELISA like sandwich method may be used without departing from the original spirit and scope of the invention.

(46) The current invention improves the sensitivity of biosensor platforms such as those outlined above. The embodied biosensor technology can assess real-time surface interactions between an antibody and an antigen associated with a disease state or environmental contaminant. The biosensor platform measures attenuation of traveling shear-horizontal surface acoustic waves (SH-SAW) caused by the accumulation of mass of bound antigens to the sensing area functionalized with a target-specific protein molecule. There are four guiding design principles for the current invention: time, limit of detection (LOD), high signal-to-noise (SNR), and portability. To achieve these design objectives, the invention is divided into two core areas of focus: capture of the analyte from solution, and the detection of the analyte via the SAW sensor.

(47) A typical SH-SAW biosensor measurement results in a steady-state phase shift due to the attenuation of the waveform. The phase shift can be correlated to the mass of antigens bound to the surface when the reaction reaches an equilibrium. Since the SH-SAW biosensor system is essentially a mass detection device, mass amplification strategies are necessary to achieve the desired fg mL.sup.−1 to pg mL.sup.−1 limits of detection when working with dilute samples. In one embodiment of the application, these biological mass amplifiers include engineered all-purpose endospores that express different epitopes and/or functional groups for bio-conjugation to whole immunoglobulins (IgG) or single-chain variable fragments (scFv). Engineered endospores have the advantage of displaying many binding sites, thereby increasing the rate of reaction. The all-purpose endospore for signal amplification is coated with an antibody-binding molecule (protein A for intact antibodies, protein L for either intact antibodies or scFv antibody fragments). ScFv's is utilized to improve the orientation of the binding site and/or enhance the binding affinity to the antigen, both of which result in an increase of the rate of reaction.

(48) In another embodiment of this application, functionalized magnetic beads expressing scFv antibodies on the surface can be utilized to function as a mass amplification technique.

(49) The rapid analysis of dilute samples in a fg mL.sup.−1 to pg mL.sup.−1 range can be challenging for even the most sensitive techniques. This is because at the very low concentration levels, there is typically a diffusional limit that restricts the rate of antigen binding. To overcome the diffusional limit, the biosensor is used in a convective-enhanced delivery modality where the sample is introduced to the surface of the sensor to overcome any diffusional limit imposed by having a dilute solution. To further enhance the signal-to-noise ratio and establish a quantitative measurement, the proposed biosensor employs a multi-reservoir system to sequentially deliver biological mass amplifiers and dilute detergent solutions thereby reducing non-specific binding and associated false positives (see FIGS. 14A-14C). When the biological mass amplifier is introduced after the analyte, the binding ratio of amplifier-to-analyte is unity, thus making the measurement quantitative.

(50) In summary, consider the design constraints and solutions realized in the microfluidic system 10 of the illustrated embodiment of the invention as described above. The constraints imposed on the design of the microfluidic chamber 14 arise from the process of the biological conjugation between the analyte 20 and the antibody 22 located on the surface of the SAW detector 12. The microfluidic chamber 14 must address the unavoidable limitations of the limit of detection of the SAW detector (LOD) and the magnitude of the diffusion coefficient. The minimum threshold mass detected by a SAW detector 12 is not less than 1 picogram. The sensitivity of the SAW detector 12 must be set as the minimal threshold above the total SNR of the microfluidic system 10 taking into consideration the total surface area of the sensing lane 16, the density of the antibody 22 located on the sensing lane 16, and the total volume of the buffer 24 of 100 microliters. The design of the microfluidic system 10 is subject to the magnitude of the diffusion coefficient of the sample 18, and subject to the fact that natural conjugation between the analyte 20 and antibody 22 requires many hours to meet the minimum threshold mass of detection in the SAW detector 12. To obtain a signal to reliably represent the actual concentration of the analyte 20 in question, a design of a microfluidic chamber 14 is needed to address the limitation of low concentration of the analyte 20, the density of the antibody 22 on the sensing lane 16 of the SAW detector 12, and the diffusion coefficient limitation.

(51) To overcome these unavoidable constraints the design of the microfluidic system 10 mixes analyte 20, such as any collected endospores or other mass amplifiers, and buffer 24 within a reservoir chamber 26, and generates a homogenous gross mixture 28. In one embodiment the endospore bearing analyte 20 and the buffer 24 are pre-loaded into the reservoir chamber 26 through a syringe 31 and sealing membrane 30, providing a foolproof loading protocol for handheld, field-portal, and disposable device. Since the typical diffusion coefficients would normally entail time domain which do not lend to the intended use of the invention as the device is meant to act as a handheld field-portable device providing near real time analytical results compared to conventional sensing techniques such as ELISA, PCR, and existing SAW techniques. The design of microfluidic system 10 uses a recirculating manifold 32 to enable the analyte 20 in a 100-microliter sample 18 to be sufficiently exposed to the antibodies 22 functionalized on sensing lane 16. In general, the analyte 20 must be positioned within 1 micrometer of the antibody 22 before capture or hybridization is possible. Recirculation of sample 18 increases the probability of the analyte 20 to fall within the hybridization range of the antibody 22 on the surface of the sensing lane 16, thereby overcoming the limitation of the diffusion coefficient. Unidirectional flow within the microfluidic chamber 14 is ensured by the use of check valve geometry or a nozzle-diffuser combination 34. A piezo pump 36 provides a convection enhanced delivery of mixture 28 to provide for uniform and controlled flow across the entire microfluidic chamber 14 based on duty-cycle and amplitude of the applied voltage (the duty-cycle is tailored to the association rate K+ and K−). The microfluidic chamber 14 incorporates a bubble trap 38 to maximize surface contact with a hydrophobic membrane 40 to release air within the mixture 28. Passive mixer 42 upstream from the SAW detector 12 enables a fine mixing of the mixture 28. A splitter-combiner 44 between the passive mixer 42 and SAW detector 12 provides for balanced distribution of mixture 28 into each of multiple lanes or channels 74 in SAW detector 12.

(52) In the illustrated embodiment, the handheld device and portable detector used in the field is characterized by: A time constraint of no more than 10-15 min to obtain results which are statistically commensurable with industry standards. A diffusion coefficient as well as capture rate (K+/K−) are constants that cannot be altered, but by the use of convection enhanced delivery through recirculation technique provided by the manifold and its propellant mechanism, the increase of kinetics of the analyte within the buffer solution is increased, thereby reducing the time domain by increasing the probability of encounter of an analyte element with the antibody at the sensing lane of the SAW. A mass enhancement technique is demonstrated experimentally by using gold nanoparticles, PHIX viruses, endospores, and or magnetic beads which reduces the limit of detection to the order of picogram to femtogram mL.sup.−1 volume. The method is demonstrates the ability of a multichambered fluidic apparatus to eliminate the need for an operator to perform multiple biochemistry steps such as mixing, conjugating, or cleaning (by use of a detergent) to remove unnecessary sedimentation of nonspecific binding particles, thereby reducing false positive results.

(53) Many alterations and modifications may be made by those having ordinary skill in the art without departing from the spirit and scope of the embodiments. Therefore, it must be understood that the illustrated embodiment has been set forth only for the purposes of example and that it should not be taken as limiting the embodiments as defined by the following embodiments and its various embodiments.

(54) Therefore, it must be understood that the illustrated embodiment has been set forth only for the purposes of example and that it should not be taken as limiting the embodiments as defined by the following claims. For example, notwithstanding the fact that the elements of a claim are set forth below in a certain combination, it must be expressly understood that the embodiments include other combinations of fewer, more or different elements, which are disclosed in above even when not initially claimed in such combinations. A teaching that two elements are combined in a claimed combination is further to be understood as also allowing for a claimed combination in which the two elements are not combined with each other, but may be used alone or combined in other combinations. The excision of any disclosed element of the embodiments is explicitly contemplated as within the scope of the embodiments.

(55) The words used in this specification to describe the various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

(56) The definitions of the words or elements of the following claims are, therefore, defined in this specification to include not only the combination of elements which are literally set forth, but all equivalent structure, material or acts for performing substantially the same function in substantially the same way to obtain substantially the same result. In this sense it is therefore contemplated that an equivalent substitution of two or more elements may be made for any one of the elements in the claims below or that a single element may be substituted for two or more elements in a claim. Although elements may be described above as acting in certain combinations and even initially claimed as such, it is to be expressly understood that one or more elements from a claimed combination can in some cases be excised from the combination and that the claimed combination may be directed to a sub combination or variation of a sub combination.

(57) Insubstantial changes from the claimed subject matter as viewed by a person with ordinary skill in the art, now known or later devised, are expressly contemplated as being equivalently within the scope of the claims. Therefore, obvious substitutions now or later known to one with ordinary skill in the art are defined to be within the scope of the defined elements.

(58) The claims are thus to be understood to include what is specifically illustrated and described above, what is conceptionally equivalent, what can be obviously substituted and also what essentially incorporates the essential idea of the embodiments.