HK1234110A1 - Portable nucleic acid analysis system and high-performance microfluidic electroactive polymer actuators - Google Patents

Description

Portable nucleic acid analysis system and high efficiency microfluidic electroactive polymer actuator

The present application claims serial No. 62/081,525 filed on 11/18/2014; serial No. 62/041,430 filed on 25/8/2014; and priority of serial number 61/979,377 submitted on day 4, month 14, 2014.

The invention is completed under the government support of national defense department advanced research project organization under contract number HR 0011-14-C-0082. The government has certain rights in the invention.

Introduction to the design reside in

Integration of sample preparations by amplification and detection in easy-to-use systems remains a significant challenge for nucleic acid diagnostics [1 ]. The FDA has approved a variety of systems as "moderately complex" devices for near-POC applications [2], including the GeneXpert platform of seepei corporation (Cepheid), the Verigene platform of Nanosphere corporation (Nanosphere), the BD Max system of BD corporation (Becton, Dickinson and Company), and the "lab-in-a-tube" disposable device of liett corporation (Liat).

Research literature is ubiquitous as an example of pathogen detection techniques and devices; however, none have been able to develop a multiple, automated, integrated "sample to result" system configured to accept a raw biological sample [3 ]. Early examples used electrophoretic separation and laser-induced fluorescence to detect the presence of pathogen DNA amplified and extracted from whole blood [4 ]. Xu et al [5] use real-time fluorescence reading during amplification to detect as few as 100 copies/. mu.l. Ferguson et al [6] detected viral RNA hybridized to PNA probes on nanostructured electrodes by electrophoresis in the presence of electrocatalytic buffer, while Lam et al [7] detected hybridization of amplified ssDNA to redox-labeled molecular probes deposited on gold electrodes. Ferguson [6] demonstrated that an LOD equal to 10TCID50 (tissue culture infectious dose) was obtained, which is 4 orders of magnitude less than clinical efficacy value. Lam 7 confirmed that an LOD of 1 bacterium/. mu.l was obtained, although a concentration of 100 CFU/. mu.l was used for the analysis on spiked urine samples. Group 2 used the lateral flow sandwich assay as the basis for their nucleic acid detection [8,9], and the Fraunhofer Institutes ivD-platform [10] used a modular platform.

The department of defense (DoD) has recognized the need for on-site portable bioanalysis for decades. Initially, this need was for identification of biological threats; however, with the advancement of personalized medicine, DoD recognizes the value of conventional health status monitoring and the accessibility of point of care (POC) diagnostics in addition to environmental monitoring. In fact, DARPA has a variety of procedures aimed at monitoring biological systems to allow rapid intervention. However, despite decades of investment, there is no commercially available instrument for processing nucleic acids at the site of need. To fill this gap, SRI has been developed and we disclose herein a portable, integrated, rapidly reconfigurable, and automated biological detection system that performs "sample entry to answer output" analysis.

Disclosure of Invention

The present invention provides devices, systems and methods for parallel detection of a set of different nucleic acid sequences by multiple sequence amplification and simultaneous hybridization reading.

In one aspect, the invention provides an automated nucleic acid analysis system comprising a sample lysis, amplification, PCR and detection module in microfluidic communication configured to perform parallel detection of different nucleic acid sequences by multiple sequence amplification and simultaneous microarray hybridization reading.

In an embodiment, the present invention provides a system, wherein:

the detection module comprises microarray detection optics comprising a microarray scanner excited with evanescent waves;

the detection module comprises an automated hybridization processor configured to provide multiple stringencies by temperature; and/or

The PCR module is configured to perform reverse transcription and PCR in a single reaction.

In an embodiment, the invention provides a system comprising an integrated microfluidic card comprising a module and an analyzer comprising a receptacle configured to receive the card, an operator configured to operate the card, and a controller configured to electronically control the operator, the operator comprising a fluidic actuator, a PCR thermal cycler, and an automated hybridization processor, and microarray detection optics.

In embodiments, the invention provides a system further comprising a reagent module configured to contain and deliver reagents to the lysis, purification, PCR, and detection modules.

In an embodiment, the present invention provides a system that is:

the method is portable: less than 1000in³And less than 10 lbs;

the method is rapid: analysis was less than 120 minutes;

multiple: simultaneously analyzing more than 50 target sequences; and/or

Automatically: no user intervention is required between sample introduction and result presentation.

In an embodiment, the present invention provides a system, wherein:

the sample comprises a protein analyte and the system is further configured to label the protein analyte with a tag comprising a nucleic acid sequence;

the anchored probe defines a sequence by its spatial position;

amplification is achieved using fewer primer pairs than the number of sequences analyzed;

the different nucleic acid sequences are of multiple species/organisms;

the PCR module comprises a metal (e.g., aluminum) PCR reaction chamber;

the microfluidic communication comprises a gas permeable membrane configured for removing gas bubbles, wherein the gas permeable membrane is below the channel layer such that the entire channel is exposed to atmospheric pressure (in a specific embodiment, the membrane spans the card as it is more easily layered than a separate sheet, although it only functions below the channel layer);

the amplification is contained entirely in a consumable (e.g., a non-open tube); and/or

Detection is based on probe sets rather than primer sets (new assays are easily constructed).

In an embodiment, the invention provides a system configured to:

amplification in a single vessel (no sample lysis);

the following sample analytes were received and processed: blood, saliva, GI samples, urine, wound swabs, spinal punctures, nasal swabs, veterinary and agricultural sources;

receiving a sample via a specimen collection vehicle or transport medium;

treating a sample volume of 1-100 ul;

are modular (modules are interchangeable to support different applications);

can be measured (by channel size and bubble removal); and/or

Are one-way or self-sealing (to prevent cross-contamination of samples).

In an embodiment, the invention provides a system comprising an integrated microfluidic card comprising a module and an analyzer comprising a housing (cartridge) and comprising a receptacle within the housing configured to accept the card, wherein the analyzer:

the cards were ligated for lysis, purification, PCT (amplification and labeling), and detection;

interaction with the sample by pressure (e.g., sample transport), magnetic field (e.g., sample mixing), temperature (e.g., amplification, stringency, hybridization), and/or light (e.g., hybridization detection); and/or

Detection is performed by coupling an evanescent wave to the sample to observe hybridization in real time and/or to determine possible base pair mismatches and kinetics of generating sequence information.

In an embodiment, the invention provides a system comprising an integrated microfluidic card (cartridge) comprising a module, wherein the card is configured to:

specific for the type of disease (e.g., respiratory disease);

specific to the type of patient (e.g., pediatric);

specific for the pathogen type (e.g., biological warfare agents);

specific to the individual (e.g., pharmacogenomics);

a unique identifier containing information specific to the patient;

for single use to maintain sterility and minimize cross-contamination;

produced using a roll-to-roll production step; and/or

From a polycarbonate chassis, a metal foil PCR chamber, an acrylic component, a breathable film material, and/or a polyurethane seal.

In embodiments, the present invention provides a system for functionally integrated microfluidic particle sorters, such as a fluorescence-activated cell sorter (FACS), configured to provide hydrodynamic and/or inertial focusing for particle or cell alignment and comprising microporous electroactive polymer (EAP) actuators configured for sorting.

EAP μ -sorters can be functionally integrated or incorporated into the particle-concentration/sorting module of the mfc system and configured to allow the system to increase the operating area (operation envelop) by concentrating large volumes of dilute particle concentrations (e.g., bacteria present in environmental samples at a few cells per ml) or sorting out selected cells from a background of many cells (e.g., sorting out activated T cells from a peripheral blood mononuclear cell population). In addition, the microporous electroactive polymer actuators are suitable for alternate applications in sorting, including cell capture, fluid mixing, and pumping, and thus can be provided, configured, and/or operated independently of the subject automated nucleic acid analysis system.

The invention also provides methods for parallel detection of different analyte nucleic acid sequences by multiplex sequence amplification and simultaneous microarray hybridization reads using the disclosed system.

In another aspect, the invention provides a high efficiency microfluidic electroactive polymer (μ EAP) actuator disposed around a fluid channel, wherein a voltage pulse applied to the actuator induces the actuator to generate a transient cross-flow between the fluid channels that deflects target particles within the fluid channel onto a new path, wherein the actuator comprises a closed-end fluid chamber, wherein one or more surfaces (e.g., walls, bottom layer, top layer) of the chamber comprise an electrode covered by an EAP layer of a dielectric elastomer.

A single uEAP actuator may be paired with a compliant chamber (i.e., a "bellows") that receives a fluid jet driven by the actuator. This configuration requires only one working actuator, but it still generates cross flow. The compliance chamber may be just one of the actuators without electronic connections, or it may be a chamber of a different geometry, as we use for a multi-channel/stage sorter.

Although primarily illustrated with solid electrodes (e.g., Indium Tin Oxide (ITO) electrodes on a glass sheet), the electrodes may also or additionally comprise a fluid, such as a conductive fluid in adjacent channels.

In another aspect, the invention provides a plurality of such actuators disposed around a fluid channel and asynchronous to each other, wherein a voltage pulse applied to the actuators induces the actuators to generate a transient cross flow between the fluid channels that deflects target particles within the fluid channel onto a new path, wherein each actuator comprises a closed-end fluid chamber, wherein the surface of the chamber is an electrode covered by an EAP layer of a dielectric elastomer.

In another aspect, the plurality of actuators is a pair of actuators disposed 180 ° out of phase with each other.

In an embodiment:

-the surfaces of the chamber contain electrodes covered by an EAP layer of dielectric elastomer;

-the fluidic channel is arranged to provide a combination of hydrodynamic focusing for horizontal alignment of the particles and inertial focusing for vertical alignment;

the new path is guided to be sorted and output;

the fluidic channel comprises a sample input channel and sorted and unsorted output channels, and a new path leading to the sorted output channel;

the fluid channel is configured for fluorescence detection, wherein after detection of the target particles, a voltage pulse is applied to the μ EAP actuator;

-the EAP layer is 1-50 (or 2-25, or 5-15 μm thick);

the elastomer is a silicone;

-the actuators are arranged to provide parallel sorting in a multi-channel device;

-the actuator is arranged to provide a multi-stage series of sorts to the plurality of outputs; and/or

The actuator is functionally integrated into the label activated particle sorter.

The invention also provides methods of making and using the actuators, such as including the step of applying voltage pulses to induce the actuators to generate transient cross-flows between the fluid channels that deflect target particles within the fluid channels onto new paths.

The invention provides in particular all combinations of the described embodiments as if each had been listed individually and specifically.

Brief description of the drawings

FIG. 1: physical construction of molecular diagnostic systems.

FIGS. 2A and B: an iMGC card.

FIG. 2C: a nasal swab input module.

FIG. 2D: and arranging TECs.

FIG. 2E: cross section of the imafc card around the PCR and detection processing module.

FIG. 2F: a glass substrate having a detection aperture, a grating, and chromium.

FIG. 2G: a side view of a glass substrate showing how light is coupled into the glass substrate.

FIG. 2H: a side view of a glass substrate showing how light propagates by total internal reflection within the glass substrate.

FIG. 2I: camera systems and arrangement of TECs in relation to the microarray.

FIG. 3: a mechanism for binding nucleic acids to a quartz frit.

FIG. 4: and controlling the volume of the breathable film.

Fig. 5A and 5B: a valve control mechanism to control the flow.

FIG. 6: projection view of the mfc card.

FIG. 7: a pneumatic system.

FIG. 8: functional block diagram of the physical hardware of the device 10.

FIG. 9: schematic EAP differential selector showing channel layout for μ EAP FAC.

FIG. 10: fringe pattern of 7- μm green fluorescent particles.

FIG. 11: phycoerythrin-labeled B cell sorting.

FIG. 12: multiple sorters are integrated into a dual channel device.

FIG. 13: a multi-sorter is integrated into a staged sorting device.

Detailed description of the embodiments and examples

The present invention provides a portable, inexpensive molecular diagnostic system that can produce results without human intervention-human intervention is only required in inputting samples and reading results. The results are also very rapid due to the various techniques described above. The system includes a method of joining disposable elements called mfcs. The method and system make it easy to set up the iMFC to run different types of tests without the need to change the hardware behind. Finally, the card itself is modular, and can be easily changed to run different types of tests.

In one aspect, the present invention provides a stand-alone system that can take a sample as input, perform a molecular diagnostic step, and display the results of the diagnostic test. Generally, this step includes (i) extraction and purification, for example, where a DNA sample is extracted from an input sample, which may be blood, tissue, urine, saliva, or other bodily fluid; (ii) amplifying and labeling, e.g., wherein a specific sequence of a sample is amplified; (iii) hybridizing; (iv) (iv) stringent washing to remove unwanted molecules that bind to the target sequence and (v) reading where the identification step is completed and the specific sequence is identified. The system generally includes a disposable element called an integrated microfluidic card (mfc) and a non-disposable base unit. The integrated microfluidic card may be coupled to the base unit through an interface scheme. Fluid flow in the mfc may be achieved by controlling valves that form part of the card structure. Each card can be designed to detect and identify a specific set of biomarkers.

In embodiments, the steps between sample input and reading are performed without any human intervention, the system does not split the sample, the system is capable of identifying at least 1000 targets, the system can identify more than 50 targets per run and/or perform all testing steps in a single system, and/or by using real-time hybridization, the system is capable of predicting results before hybridization is complete and is capable of providing information related to target concentration.

Fig. 1 shows the entire system 10. The system includes a cell substrate 20, a cell lid 30, and a disposable integrated microfluidic card (mfc) 40. The design of the card is modular, such that it can be customized to accept various forms of sample input, including but not limited to blood drops, nasal swabs, sputum, etc., or customized to perform different tests. Generally, the testing process begins by first selecting the appropriate card, placing the sample in the input chamber of the card, placing the card in the cell base, closing the lid and selecting the appropriate software run. No human intervention is required once the testing process is initiated. The results may be displayed on a screen integrated as part of the system, or they may be transmitted to an external device such as a computer or smartphone. After the test is complete, the card may be processed and a different card may be selected for the next test. The order of these steps may be varied as desired.

A disposable integrated microfluidic card. The card generally combines multiple functions on a single card, such as lysis, purification, amplification, labeling and detection in a typical commercially available system, with multiple instruments to accomplish these functions. We have achieved functional integration by integrating multiple techniques and features for bubble removal and accurate measurement, integration of evanescent field imaging systems, selection of reagents and chemicals in various steps, rapid amplification and real-time hybridization, etc. The incorporation of multiple functions into one card enables CLIA exemption (CLIA driver). Fig. 2A shows a perspective view of the card and fig. 2B shows a plan view. Fig. 2B also shows an area (shown in phantom) in which various functions may be present within the card. For example, block 100 may be a lysis block, block 110 may be a purification block, block 120 may be where a Polymerase Chain Reaction (PCR) process occurs, and block 130 may be where a detection occurs. The fluid containing the test sample flows in the above-described order from one patch to another through channels incorporated in the card. The fluid flow through these passages from one block to the other is controlled by a miniature valve, which can be opened or closed depending on the pressure applied at the valve.

And (4) cracking the block. Sample input may also occur in the lysis block, fig. 2A and 2B, block 100. Various methods can be used to input the sample, such as the use of blood drops and nasal swabs. In one embodiment, the card directly accepts the tool for collecting the sample. Generally, in commercially available systems, the tool used to collect the sample does not directly engage the test instrument. By allowing the tool to directly engage the test instrument, sources of contamination and user error may be eliminated. Because the card is a module, the split block can be changed to accept various input methods. Referring to fig. 2A and 2B, element 102 may be an input chamber that receives a sample, such as a drop of blood. Fig. 2C shows the advantage of modularity, wherein the lysis block is configured to accept a nasal swab 250 as an input. For both cases where a drop of blood or a nasal swab is used, the back design of the card 40 may be the same; only the input modules may be different.

In addition to accommodating various input methods, the lysis module may be configured to perform different types of lysis, such as mechanical, chemical, or other types. Referring to fig. 2A and 2B, the lysis module is shown with a bead mill 104 comprising a bead chamber filled with beads and a small electric motor fixed at the top of the bead chamber which causes the beads to collide with each other. Cells located between the colliding beads lyse, releasing their contents into the lysis buffer-an example of mechanical lysis. Other modules may be provided to perform other forms of lysis, and these modules may have different or fewer chambers or sub-modules than shown in fig. 2A and 2B. For example, a module capable of chemical lysis may have another chamber in place of a bead mill, where the chemicals (possibly stored in a reagent module and entered the chamber at the appropriate time) may be mixed with the lysis buffer containing the sample. Thus, different types of cracking may accommodate the same basic design of the card.

To initiate the lysis process, lysis buffer solution stored in lysis buffer well 142 in reagent block 140 is passed into chamber 108. The method of controlling fluid flow in a card can be achieved by controlling a microvalve. The chamber 108 may have an air-permeable breach at the top, such that when liquid contacts the breach, it is sealed off by air due to the pressure difference between the chamber and the atmosphere, such that air in the chamber flows out as it fills. The slit may be made of various materials, such as Teflon (Teflon). When the chamber 108 is filled, a valve between it and the input chamber 102 may be opened so that the buffer solution mixes with the input sample. Once the lysis buffer and input sample are mixed, another valve between the input chamber 102 and the bead mill 104 is opened to allow the mixture to enter the bead mill. The electric motor at the top of the bead mill is then turned on for a specified time, after which the valve between the bead mill 104 and the chamber 106 is opened and the solution now flows into the chamber 106. The chamber 106 may be pre-filled with a reagent, such as guanidine hydrochloride, where it is mixed with the lysed solution. After mixing with the reagent is complete, a valve between the chamber 106 and the purification block 110 may be opened to direct the mixture to the purification block. Hydrochloric acid causes the DNA in the sample to bind to the silica-based structure in the purification block, thereby assisting the purification process.

Configurations that provide the ability to perform different types of lysis, accept various input methods, and accept practical tools for input methods are advantageous features that may be synergistically combined in the device 10.

And (5) purifying the block. When the valve between the chamber 106 and the purification block is opened, the solution comes into contact with the quartz frit placed in the purification block. The frit and frit-containing structures are shown in fig. 2A and 2B as 112 and 114, respectively. The flow of the solution between the frits is shown in fig. 3. The figure shows a cross section of the card around the frit 112. The frit may be in the form of a channel 410, which may be sandwiched between the base layer 400 and the cap layer 430. The base layer and the cap layer form part of the frit-containing structure 114. The arrows indicate fluid flow within the channel 410. The solution containing the nucleic acid in the solution containing guanidine hydrochloride from chamber 106 will bind to the frit and flow in the direction of the arrows. The flow of solution after it has flowed through the frit will be directed by the microvalve to flow into the residual chamber or to further processing. Thus, DNA in the solution binds to the frit, but other components of the solution that do not bind to the frit, such as proteins and lipids, flow through the frit and can be directed through the channels into the residual collection chamber 114. After the DNA binding step, a washing step is performed. The ethanol contained in the ethanol reservoir 146 allows flow through the frit to perform a washing step to wash away unwanted lysate components that may still be bound to the DNA. The ethanol was then allowed to dry. The next step in the purification process is to wash the elution buffer stored in the elution buffer well 148 over a frit. This step decouples the nucleic acid from the frit. The solution is now allowed to enter the PCR processing block 120. In this step, the valve leading to the further processing step (PCR process) is opened, but the valve leading to the residual chamber is closed.

And (3) PCR blocks. After purification of the block, Polymerase Chain Reaction (PCR) can preferably be performed with system modifications as described below. In a PCR block, indicated by 120, and PCR may be performed in multiple steps as shown in fig. 2A and 2B. After the elution wash from the purification step is complete, the purified nucleic acid-containing solution is allowed to flow into the PCR main mix chamber 122. Here, the purified solution is mixed with a lyophilized (freeze-dried) enzyme, which is carried within the main mixing chamber structure. These enzymes are required for further amplification steps to occur in the processing strand. The main mixing chamber may contain an aperture, wherein the volume of solution filling the aperture may be controlled, allowing for precise volumes of solution, and avoiding or eliminating air bubbles. The precise volumes in the various processing stages achieve the accuracy of the results.

After mixing with the enzyme is complete, the solution is flowed into the PCR primer chamber 124 where it is mixed with the lyophilized primers. The primer chamber may also be a well in which the volume is precisely controlled in addition to avoiding or eliminating air bubbles trapped in the solution. In some cases it may be preferable to separate the mixing step comprising mixing the master mix and the primers, as this allows the use of commonly used master mix modules and reduces the overall cost of the device. In addition, the division into 2 steps also allows for rapid deployment of the kit to test for emerging problems, e.g., multiple cards may be made with different primers, which can be used to check for the presence of different target molecules. The process of lyophilization of the primers (oligonucleotides) is rapid and simple and can be performed in a laboratory outside the device 10. Thus, by separating 2 mixing steps, the card can be altered to test for different substances, although if preferred, the mixing process can also be performed in one step.

After mixing with the primers is complete, the solution flows to the PCR chamber 126. In a different approach than conventional commercially available approaches (where the PCR chamber may be made of non-metallic materials), the PCR chamber in the mfc card may be made of a passivating metal, such as, but not limited to, aluminum. The use of metals, particularly aluminum, is preferred because it allows for rapid control of the temperature of the solution within the aluminum chamber. This rapid control is achieved by bringing the top and bottom of the aluminum chamber into intimate contact with a thermoelectric cooler (TEC). The close contact between the PCR chamber and the TEC is obtained by placing 2 TECs above or below the PCR chamber. The TEC on the PCR chamber is located on the lid 30 of the device 10 within the TEC housing unit 200 (fig. 1). The shape of the face of the TEC 210 matches the shape of the PCR chamber 126 so that when the lid is closed, the TEC face can sit directly on the upper surface of the PCR chamber. The TEC under the PCR chamber is simply placed within the cell substrate 20. FIG. 2D shows how the PCR chamber is sandwiched between 2 TECs 220 (with TEC face 210) and 230 (with TEC face 240, not visible in the figure). Thus, this arrangement of TEC with the use of an aluminum PCR chamber enables rapid temperature cycling of the PCR mixture. It has been found by measurement that this combination allows a temperature increase of >15 ℃/s with an accuracy of ± 1 ℃. This configuration then directly results in a reduction of the time between sample input and result output. In addition to the advantages obtained in rapid temperature control of the solution due to the use of passivated aluminium, another advantage is achieved in that less energy is required to heat and cool the solution than is required for a PCR chamber using non-metallic materials. Less energy translates directly into less overall energy required to operate the device, enabling optional battery operation and field portability.

Another aspect of the PCR chamber 126 is now explained with reference to fig. 2E, which shows a cross section of the card around the PCR chamber and the detection block 130. To preserve the concentration of the solution, trapped air within the PCR chamber should be avoided or minimized when performing the PCR process. The top and bottom section PCR chambers may be made of passivated aluminum; thus, breathable films may not be used herein. Thus, an output channel 250 from the PCR chamber is provided which leads to a reservoir 255 and is open to atmosphere via another channel 257. Thus, as the chamber 126 fills with solution, air is pushed out and flows to the atmosphere. Upon filling the chamber 126, the valve in the passage 260 may be closed. During the filling process, the channel 250 and reservoir 255 may also be filled with a solution, which prevents air from returning to the chamber 126. At the same time, no backflow from channel 250 or reservoir 255 occurs as the solution is driven by a constant pressure of 6 psi. After filling the chamber, the PCR process is started. At the end of the PCR process, the desired target sequence is amplified and labeled for detection in the detection block.

Detection Block-general description. After the PCR process is complete, referring to fig. 2E, the valve 262 in the channel 260 can be opened and the solution containing the amplified components (amplicons) in the PCR chamber 126 can flow into the mixing chamber 275. Thus, the solution is mixed with hybridization buffer that can be stored in the hybridization buffer well 150 in the reagent block. The solution from the hybridization buffer is measured before it is mixed with the PCR product. After the mixing process in the mixing chamber 275 is complete, the solution is flowed into the detection chamber 325 through the channel 330. The detection chamber 325, the mixing chamber 275, and the channels for the input and output of the solution from the chambers all form part of the detection block 130 shown in FIG. 2B. Next, within the detection chamber, PCR product mixed with hybridization buffer can be flowed onto the DNA microarray. This occurs in well 317, shown in FIG. 2F, where the well holding the solution and microarray 315 is placed at the bottom of the well. Thus, for this configuration, the solution is located on top of the microarray. FIG. 2F shows the arrangement of a DNA microarray 315 and explains how light, which forms part of the optical system for reading the microarray, is coupled into the microarray. The DNA microarray may contain a plurality of probes arranged in a grid pattern on the glass substrate 310. Each probe may contain one strand of DNA (or RNA) and is used to detect the presence of a nucleotide sequence in a sample solution that is complementary to the sequence in the probe. As described above, these probes are placed in a grid pattern on top of the glass substrate 310. Light is coupled into the glass substrate through a grating 305 etched into the glass.

Referring to fig. 2G, a grating is shown along incident light 306. The incident light 306 may have a particular wavelength. Due to the presence of the grating, incident light can propagate in multiple directions; some light may travel to the right as shown at 307 and some light may travel at a particular angle as shown at 308 and 309. Light may propagate at other steeper angles relative to light 307, but they are ignored herein because the light intensity at steeper angles tends to be lower. The angle of light propagation (for light that does not propagate in a straight line) is determined by well-known grating equations. The angle of light 308 and 309 can be adjusted by adjusting the wavelength of incident light 306 and patterning the grating. Next, in order to couple light into the glass substrate, a process of total internal reflection is used. This concept is illustrated in fig. 2H. In the figure, light 307 traveling straight and light 309 traveling at an angle are ignored. Light 308 is shown as a beam of light shown by the size of grating 305. Thus, the beam of light 308 is shown by a solid line 308L emanating from the left edge of the grating and a dashed line 308R emanating from the right edge of the grating. Solid and dashed lines are used to distinguish 2 edges of the beam; no other differences were shown. Depending on the angle of the beam 308 and the refractive indices of the glass substrate and the surrounding environment (substantially air), total internal reflection may be established at the upper surface of the glass substrate region 1 labeled R1. This reflected light may reach the lower surface of the glass substrate and may again be totally internally reflected at region 2 (labeled R2). Thus, light may be directed beneath microarray 315 after detection wells 317, despite total internal reflection. As previously described, microarray 316 may be located at the bottom of monitoring well 317. Now, light is directed to the location of the microarray, creating another phenomenon known as evanescent fields to excite photosensitive molecules in the DNA microarray. In the known optical element, at the boundary where total internal reflection occurs, an evanescent field is established on the other side of the boundary. These evanescent fields are near field phenomena and drop exponentially in intensity away from the boundary. However, very close to the boundary, the evanescent field is able to excite the photosensitive molecules and since the solution in the detection well 317 is located at or near the boundary, detection methods using evanescent fields become possible. Returning to FIG. 2H, it can be seen that the detection wells are located within the sealing layer 320; the sealing layer is made of chromium. The chrome layer is also shown in FIG. 2F around the test hole on all four sides. Chromium is included as part of the design to reduce or eliminate scattered light emitted from the vicinity of the detection aperture. The possibility of false light is further removed by coupling a black plastic sheet 327 over the detection wells. Other materials besides plastic may also be used.

The grating design and subsequent angle of light 308, the thickness of the glass substrate, the refractive index of the glass substrate, the distance between the grating and the detection hole are parameters that we have optimized for the device and provide synergistic functionality. Additionally, the angle of light 308 may be selected such that it is totally internally reflected not only from regions such as R1 and R2 (substantially from the glass-air boundary), but also from the glass-solution boundary of the detection well. The reflectance of the solution in the detection well was about 1.33, while the reflectance of air was about 1. The grating pitch, grating material and substrate ratio can be varied to use different wavelength lasers. In one embodiment, 633nm wavelength light is used in conjunction with a 195nm pitch 150nm silicon nitride grating on a 750 micron fused silica substrate. The light is coupled into the substrate with a 10 ° divergence. The distance between the grating and the microarray is selected such that the microarray is located at an integer distance corresponding to total internal reflection. All of the above specific parameters may be adjusted as desired. For example, other wavelengths of light may be used, which then require a different grating pitch than described above.

A TEC can be placed over the detection well 317 to control the solution temperature. Finally, a CCD camera 360 can be placed at the bottom of the microarray so that a one-to-one image of the microarray can be formed on the camera detector. The camera system takes a picture of the solution on the microarray. The photograph shows the area within the microarray that may be fluorescing. This information is used in the authentication and detection process.

The present invention utilizes a synergistic combination of such improvements and adjustments to perform the detection function.

Detection block-quality control. In addition to identifying target sequences within a sample, microarrays can be used to ensure that steps (lysis, purification, mixing of enzymes and primers, etc.) occur as described above. Labels can be added at each step and the microarray can be optically tested for the presence or absence of labels. Thus, by analyzing the presence or absence of the marker, quality control can be achieved to identify whether or not the test may not have performed properly.

Detection block-total amplification. The device 10 can be used to avoid sample lysis for testing various nucleic acid sequences. Sample lysis is commonly used in commercial systems; which requires that the sample be divided into a plurality of samples, wherein each divided sample can be tested for a specific sequence. Since the original sample is split into multiple samples, this method reduces the detection limit by a factor equal to the number of sample splits. Instead of using sample splitting, we perform a process called whole amplification, whereby variations within a bacterial or viral species can be identified without splitting the sample. This type of testing becomes possible because some portions of the DNA of the variants within a species may be small or similar. Where certain sequences are known to be present, primers can then be designed to identify the variants.

In general, PCR is limited to about 20 different primer sets, which limits the number of targets that can be detected in one commercially available system. Our system overcomes this limitation by full amplification, where the PCR primer set targets DNA sequences that are common among many organisms, while the region between the primers contains DNA sequences that are highly variable between organisms. The full amplification method allows differentiation of sample types on a microarray, where high multiplexing is possible. For example, a conserved region of the coronavirus polymerase gene allows for amplification of 6 different coronavirus serotypes with a single primer set, and each serotype can be distinguished over a microarray by analysis of the variable region amplified between the primer sets.

One advantage of full amplification is that fewer primers are required than typical commercially available devices for performing PCR. The use of more than 20 primers can lead to a phenomenon known as "primer dimer" in which the primer molecules hybridize to each other due to the strand of complementary bases in the primers, whereas our full amplification process allows the use of fewer primers, thus resulting in an advantageous configuration.

Detection of blocks-real time hybridization. In another advantageous configuration, a real-time hybridization method is implemented within the device 10, which results in a reduction in the time between sample input and result output. In a typical commercial method, hybridization is allowed to continue until a stable concentration of the sample or samples being tested is reached. In contrast, in the device 10, a real-time method is performed in which the concentration of one or more samples is repeatedly estimated during a period in which the concentration of such one or more samples may rapidly rise after hybridization begins. This technique is based on the observation that the signal intensity from the CCD camera may be related to the concentration of the dynamic curve equation, supra. Using a dynamic model, the increase in fluorescence signal with respect to time can be monitored. The signal may be modeled or fitted to an exponential form from which the time constant may be estimated. With an estimate of the time constant, the analyte concentration can be estimated.

Detection block-using a multi-stage temperature control. In a typical commercial hybridization process, after hybridization is complete, stringent washes are performed to wash away unbound probes. In general, stringent washes are performed by varying the salt concentration, however in device 10 a similar effect can be achieved by varying the sample temperature using TEC while maintaining a constant salt concentration. Thus, after hybridization is complete and an initial set of pictures is taken, the temperature of the TEC can be raised so that some of the unbound molecules can be removed. This allows a preferred way of checking the results, since it is easier to change the temperature of the solution than to change the salt concentration of the hybridization buffer.

And (4) volume control. The testing process requires a precise volume, which can be destroyed by the presence of bubbles in the fill chamber. In typical commercially available systems, various devices such as peristaltic pumps are used, but the addition of these devices increases the complexity and cost of the device. Our device provides volume control that achieves high precision and accuracy (preferably 10% or less) and is cost effective to implement.

Fig. 4 shows a cross section of the filling chamber 450 and the structures surrounding the filling chamber. The fill chamber may be located on top of the bottom layer 490. The side of the chamber is shown as 460. Fluid may enter the chamber along arrows 480. The output of the filling chamber may be marked with dashed arrow 495. Element 470 may be a valve; if it is off, no flow occurs along dashed arrow 495. The vented membrane 440 may be secured on top of the filling chamber as shown. Closing the valve, fluid may flow in the direction of 480 into the fill chamber 450. The air permeable membrane allows trapped air to vent and as the filling chamber fills, all air is vented. The vented membrane does not return any air due to the pressure differential between the chamber and the atmosphere; thus, by closing the valve 470, a precise amount of fluid can be collected in the fill chamber. The accuracy of the volume within the fill chamber may be determined by the accuracy of the chamber dimensions, and well known techniques may be used to control the chamber dimensions, such as, but not limited to, processing and etching. The breathable film may be made of a material such as, but not limited to, microporous polypropylene. Thus, with a precisely made combination of chamber and gas permeable membrane, precise and accurate volume control can be achieved.

The fill chamber and membrane are located together in various locations within the mfc card. Thus, for example, a fill chamber with a gas permeable membrane may be used for the PCR master mix chamber 122 and the PCR primer chamber 124. In these two specific positions, the lyophilized compound can be located as a pellet within the chamber; thus, rehydration processes within these chambers may be occurring in a volume controlled environment.

And controlling the valve. Fig. 5A and B show how the valves and fluid flow within the mfc card are controlled, including fluid flow channels 520 and 540 and flexible membrane 530. FIG. 5A shows 2 additional arrows 550 to show that fluid may flow through fluid flow channel 520. Fluid may be pumped through the system at a particular pressure, such as 6 psi. If the membrane 530 is not deformed, as shown in FIG. 5A, the channels 520 may allow fluid to flow freely. However, if a higher pressure is applied to the gas flow channels, such as 18psi, the membrane may deform, as shown in FIG. 5B, which may effectively block flow through the channels 520. Thus, by controlling the pressure on either side of the membrane, the flow through the fluid channel can be controlled. The following describes a pneumatic system applying different pressures.

An iMFC layer. The aforementioned iMFC cards can be disposable and can be made from inexpensive materials such as polycarbonate, acrylic, and polyethylene terephthalate. The card may be considered a "motherboard" in which different molecules may be accommodated, e.g., the card may be designed with a variety of different input types to accommodate different input methods. Not only can the card accommodate various modules, the channel configuration can also be modified to accommodate different diagnostic tests or to add or subtract steps from diagnostic tests. Thus, each card can be designed to accommodate a particular test or group of tests without the need to change the underlying hardware. Each card may be made of one or more layers. One of the primary functions of the card is to route fluid from one location to another at the appropriate time through the airflow channel and fluid flow channel system built into the card. Other functions include, but are not limited to, measuring flow and attesting to temperature controlled environments.

Fig. 6 shows a composite "projected" image of all layers of the mfc card. Generally, the card has one or more channels, such as 600, so that fluid can flow from one location to another in these channels. The channel may be formed by cutting a slot into the card interior laminate material. Additionally, the card may also have one or more valves, such as 630, and one or more channels or reservoirs, such as 620, in which the volume may be measured. Some layers or portions of layers may be composed of breathable films. The card may contain multiple ports, such as 610. The port may form an interface between the hardware and the card. These ports are used to apply either a driving pressure (to drive a fluid) or a valve pressure (to drive a valve). The location of these ports can remain the same for the card, which prevents any need to change the hardware behind. However, the card may be designed in any convenient manner, and the functions on the card may be set in any convenient manner. This aspect makes the card versatile in that it is designed for various tests without the need to change the underlying hardware.

A pneumatic system. The pneumatic system (fig. 7) is responsible for regulating the flow within the card. The pneumatic system may have a pump that may be located within the base of the cell. Such a pump compresses the air in the accumulator to a desired pressure, such as 16 psi. The figure also shows a feedback loop from the accumulator to the pump so that the pressure in the accumulator remains constant. One path leads from the accumulator to the regulator, where the higher pressure in the accumulator is regulated down to a lower pressure, such as 6 psi. This lower pressure is used to drive the fluid within the entire system. A solenoid valve may be included at the output of the regulator to turn on the fluid drive. Another path from the accumulator may be controlled by one or more solenoid valves to open or close the valves. Thus, the higher pressure of the accumulator (16psi) can be used to control the valve. As shown in the figure, each solenoid valve may control one or more valves. Fig. 5A and 5B show how fluid flow can be controlled by the design of the fluid and air flow channels. In the context of fig. 7, the fluid channel may be connected to the "drive" output and the gas flow channel may be connected to one of the "valve" outputs.

The pneumatic system may be part of hardware; thus, it may not be easy to change some of the paths used to provide pressure to the fluid flow or valve control. However, to achieve flexibility in card design, only ports are needed at the same location; these ports are responsible for providing pressure to the fluid drive passage or to the air flow passage to drive the valve. These ports are shown as 610 in fig. 6. Thus, by requiring the card to have ports at the same location, the same hardware unit can be used, but the card itself can be designed for different purposes.

Hardware. As shown in fig. 1, the described test system may be constructed as a disposable cartridge comprising a unit base 20 and a unit cover 30. Various functions and capabilities can be physically configured within the base and lid. Fig. 8 shows a functional block diagram of the hardware. The hardware may include a processor and a power source such as a battery. The processor may engage other elements such as a TEC, electric motor, optical system, pneumatic system, display, and communication system. With respect to display systems, the device 10 may have a screen that may display information or results. With respect to the communication system, the unit 10 may interface with an external computer by any suitable communication method, such as Ethernet and Bluetooth. These display and communication subsystems may be implemented using well-known techniques. Fig. 8 also shows that the card may mechanically engage some of the subsystems, such as the TEC, electric motor, etc. These bonds are shown by dashed lines.

Embodiments of the invention include:

1. a modular diagnostic device, system or method that detects a large set (>6, >10, >20 or >50) of nucleic acid sequences without any human intervention to conduct the testing process except for the purpose of injecting or inserting a sample and reading the results.

2. A device, system or method as described herein, wherein the lysis, purification, PCR and hybridization steps are integrated into one system, and wherein these steps are performed in a disposable card.

3. The devices, systems, or methods described herein, wherein a disposable card may contain a module, such that by changing the module, the card may be configured for different tests.

4. A device, system, or method as described herein, wherein an analyte can be detected in a sample from a variety of sources, including, for example: blood, saliva, GI sample, urine, wound swab, spinal puncture, nasal swab, veterinary and agricultural sources.

5. A device, system, or method as described herein, wherein the means for collecting the sample can be directly input into the card.

6. The devices, systems, or methods described herein, wherein the amplification step of the PCR process is performed by a process called whole amplification, rather than sample splitting.

7. A device, system or method as described herein, wherein the detecting step uses a microarray.

8. A device, system, or method as described herein, wherein the microarray includes a control that verifies that each step of the testing process is occurring properly.

9. The devices, systems, or methods described herein, wherein the detecting step uses a real-time hybridization method that predicts the concentration of the sample based on a dynamic model, wherein the read time is reduced.

10. The devices, systems, or methods described herein, wherein the PCR process is performed in a passivated metal plating chamber, such that the temperature within the chamber can be rapidly controlled by placing the metal plating chamber near or in contact with the TEC, preferably wherein the metal used is aluminum.

11. A device, system, or method described herein, wherein the volume of the solution is measured by using a channel or chamber coupled to a gas-permeable membrane, such that upon filling the channel or chamber, air is vented out of the gas-permeable membrane; thus, errors due to air in the measured volume can be minimized or eliminated.

12. A device, system, or method as described herein, wherein a pneumatic system controls fluid flow by applying different pressures to drive a fluid and control a valve.

Detailed description of other embodiments and examples

Portable systems are disclosed that can analyze nucleic acid sequence content in a variety of samples. The system is capable of taking multiple raw samples (diagnostic or environmental) and performing nucleic acid extraction, purification, amplification, labeling and sequence analysis by microarray in separate units. The system is automated and rapid to allow on-site analysis of samples by users who are not skilled laboratory technicians.

In an embodiment: the system includes (1) a reusable hardware platform and (2) a consumable integrated microfluidic card (iMFC) that determines the assay to be performed;

the system is small (<150，<250, or 400in³) Light weight (1)<3, 5 or 10lb), fast, and intuitive to use.

Hardware controls can be reused and provide pneumatics, pressure regulation, temperature control, laser control and final microarray imaging;

the spent mfc contains cards that perform sample lysis, purification, PCR, and detection and contain all the required dry reagents; and/or

The mfc also contains a reagent storage element that holds all of the liquid reagent separately so as not to compromise the integrity of the dry reagent.

In an embodiment, the system provides:

easy to use: easy to operate for field use by unskilled operators and as a result easy to understand; set to clinical laboratory improvement modification (CLIA) -exempt certification; and/or the ability to provide sample input to result output without user intervention.

Configuration: the iMFC is a module that can store the main iMFC and connect separate, fast-producing, lower cost modules that define the end-product test functionality; since the primary mfc remains the same, the system provides for the immediate development of new trials as new threats emerge.

Test flexibility; sample type (from hardy spores to mammalian cells that are prone to damage) and/or analyte type (DNA, RNA, and protein).

Manufacturability and cost: the iMFC and module components can be manufactured using low cost injection molding or advanced manufacturing laser conversion processes; high speed laser conversion and precision lamination enable fabrication of iMFCs with patterns as small as 125 μm and tolerances of less than 50 μm at production rates approaching 50 feet per minute; the combination of old injection molding techniques and advanced laser conversion techniques allows complex disposable cartridges to be produced in batches of less than $ 10/card.

In embodiments, our system is fully automated from raw sample input to result output, and/or can be configured to allow multiple analysis types.

In embodiments, our system provides a portable bioanalytical platform to detect nucleic acids, typically DNA or RNA, such as microbial, typically bacterial, viral or fungal detection, and health monitoring by mRNA and protein detection, as well as cell selection and concentration. In an embodiment, the system consists of 2 elements: (1) a reusable hardware platform and (2) a consumable integrated microfluidic card (mfc) that determines the assay to be performed.

In embodiments, our system proves adaptable to a variety of applications:

and (5) detecting a bacterial reagent. Our DNA detection mfc operates in a similar manner to a laboratory procedure: lysis, DNA purification, DNA amplification and labeling, and hybridization. Our microfluidic methods of each step are highlighted below.

Lysis was accomplished using silica beads and a low cost disposable electric motor for robust lysis of sample types from hardy spores to mammalian cells.

We purified DNA by binding it to a quartz frit, washing away impurities, and eluting in Polymerase Chain Reaction (PCR), i.e., a solution. Our DNA purification module and process allows elution of high DNA concentrations in the first 6 μ Ι within about 5 minutes without user intervention. In contrast, the laboratory platform method for spore lysis and DNA purification takes 30minutes over 7 steps and requires laboratory centrifugation.

We performed DNA amplification in an aluminum-walled chamber between 2 custom thermoelectric coolers (TECs). The TEC and aluminum chamber allow for rapid heat transfer between the TEC and PCR mixture. We have demonstrated PCR duplexes of two genes (AGG and STX2) associated with the E.coli O104: H4 pathogen in 12 minutes and have detected as few as 10 genomic copies.

We used custom DNA microarrays and optical systems to hybridize DNA. The light is coupled to the glass plate using a grating and remains confined to total internal reflection. The evanescent wave on the surface is used to excite target sequences that hybridize to their complements on the microarray surface. The use of evanescent waves allows us to observe hybridization in real time. Custom photo relays and CCDs were used to image the microarray surface.

We can validate multiple tests for multiple potential biological warfare agents and establish Receiver Operator Characteristic (ROC) curves for our test and hardware platform.

And (3) detecting the virus RNA. The same technique we used to detect viral RNA as we performed for DNA detection, except we used a single reverse transcription/PCR mixture. For influenza viruses H3N2 and H1N1, we have demonstrated single master mix reverse transcription and PCR in less than 30 minutes. The field portability capability can directly utilize information obtained from the prediction program of DARPA and allow early detection of viral population mutations that result in potential pandemics in domestic herds.

And (3) detecting mRNA. We can use the same software to address mRNA analysis, update the mfc to allow mRNA capture using poly-T beads and image the microarray in real time to capture dynamic data. The use of dynamic measurements allows us to determine the concentration of each analyte just before equilibrium is reached, thereby reducing the hybridization time for general driver gene expression analysis. Our ability to rapidly analyze blood sample mRNA at a desired location utilizes DARPA invested in predictive health and Disease programs.

And (4) detecting the protein. We can transduce protein-binding events into nucleic acid reads, thus allowing our same platform the ability to test nucleic acids and proteins simultaneously. Plasma protein concentrations indicate a healthy state or environmental exposure. We have developed a 4-host-responsive protein panel, which indicates ionizing radiation exposure; similar panels for other environmental contact diagnostics may also be integrated into the platform. We used the same bead capture microfluidic cartridge as the mRNA detection to capture protein analytes on the beads. Instead of poly-T oligonucleotides, beads were functionalized with antibodies directed against specific protein analytes. Secondary antibodies labeled with oligonucleotides were used as reporter molecules for conventional immunoassays. Once the sandwich assay has been subjected to stringent washing, the same module as for DNA detection is used to amplify, label and hybridize the reporter oligonucleotide. We refer to this method as "microsphere-immuno-PCR" (MSiPCR).

Cell selection and concentration. Our front-end module allows specific cell selection and concentration using a microfluidic Electroactive (EAP) polymer cell sorter, similar to a laboratory-scale Fluorescence Activated Cell Sorter (FACS). This module increases the system operating area by concentrating large volumes of dilute cell concentrate (some bacterial cells/ml) or sorting out selected cells from a background of many cells (only activated T cells from total peripheral blood mononuclear cells). In this module, we use hydrodynamic and/or inertial focusing to align cells, and then use EAP actuators to sort based on fluorescence triggering. We have demonstrated the potential of this technique to sort at speeds in excess of 25000 cells/second.

Baseline hand-held analyzer

The hardware platform provides all the necessary hardware actuation required to support the mfc processing of the microbial DNA sample. The user interacts with the hardware platform through the computer USB port. Once the mfc is inserted into the hardware and the lid is closed, the user selects a particular process script for the desired assay. After the script is initiated, the hand-held device runs without user interaction until completion. During operation, images are transferred from the handheld device to the host PC where they are analyzed. After the run is complete, the analysis and results will be presented on a screen for review by the user.

The hardware is designed to perform cell lysis, purification, amplification and detection in a small portable device, preferably using less than 1ft³Preferably less than 200in³. In one embodiment, the hardware dimensions are 4.75in deep by 6.25in wide by 5in high, for a total volume of 148in³。

On the mfc, the membrane valve and liquid fluid flow were controlled by positive pressure. The starting subsystem is centralized on a custom acrylic manifold that connects all the starting components in the Hand Held Analyzer (HHA) and routes the outputs of these components to input ports on the mfc. Such a manifold contains an accumulator to maintain a volume of air at a prescribed pressure (18psi) suitable for microfluidic valve membrane actuation on the mfc. A single solenoid is used to trigger the drive pressure to the mfc at any time during the test of the processing device. Such a drive pressure port is routed through a pressure regulator (fixed into the integrated manifold) so that the drive pressure can be adjusted in the range of 0-10psi for any given HHA.

Table 1 shows the functional components in the baseline system and their purpose and implementation. Hardware supporting amplification (PCR) and detection modules is described in detail below as part of the baseline mfc specification. The second column lists the purpose of the functional components and the last column lists our technical approach to implementing the required capabilities implemented on a hardware platform.

Table 1.

Baseline iMFC and iMFC modules

The spent mfc contains (1) a card to perform sample lysis, purification, PCR, and detection and to hold all required dry reagents and (2) a separate holding reagent storage element to not damage the dry reagent integrity. Since the iMFC is designed in modular mode-i.e., the application specific modules are assembled on a generic card-the iMFC can be easily and quickly developed for new applications by simply interchanging modules. This versatility feature eliminates the need to redesign, develop and manufacture new cards, the most complex components. Once manufactured, the same card can be used for a wide range of applications, thereby reducing cost and development time.

A microfluidic card. The card uses positive pressure driven flow and consists of three functional layers containing (1) a fluid channel, (2)22 membrane valves to control fluid flow, and (3) a slit to remove bubbles from the fluid channel. These functional layers together comprise 9 laminated layers, around which are 2 injection moulded parts. 7 modules pre-fixed card, 4 on the upper surface (lysis module, purification filter, PCR master mix chamber, and primer chamber) and 3 on the lower surface (PCR chamber, stir bar mix chamber, and detection chamber). Fixed to the detection chamber is an optical waveguide chip containing a DNA microarray to sense the target of interest.

And (4) reagent blocks. When the user initiates the procedure, an inflatable bladder in the handheld hardware presses the reagent block onto the sharps to penetrate the foil seal, which in turn releases the liquid reagent. Using positive pressure to drive flow, air enters the chambers in the reagent block and drives the fluid reagent through the output through-holes and into the card. A compressible gasket on the card prevents fluid or air leakage at the reagent block-card interface. Table 2 lists the fluids that can be stored in the reagent block for DNA analysis. Cards designed for different applications (mRNA or protein analysis) will have different reagents. The reagent block also includes a waste reservoir containing an absorbent material to collect the reagent flowing through the card. The reagent block preferably contains all of the liquid reagents required for a single prepackaged format for biological assay analysis. The table lists the reagents currently used for DNA analysis. Buffer designation together with the purpose and precise chemical formulation of the buffer. The reagent block is a general purpose and the reagents required will vary depending on the biological assay being performed by the iMFC. Our technical approach is to use the same package, but filled with different reagents to support the new assay capabilities of RNA virus detection, mRNA detection and protein detection.

Table 2.

And (4) cracking. After releasing the liquid from the reagent cake, the next step in the automated procedure is lysis. Lysis module, which can handle even spore samples, consisting of 3 chambers: a sample chamber, a bead milling chamber (for spore lysis), and a binder chamber. After the lysis buffer from the reagent block flows into the sample chamber, the electric motor is turned on to mix the lysis buffer with the sample. The mixed sample then flowed into the bead milling chamber, where the second electric motor was run for 3 minutes to stir the glass beads and lyse the spore sample by bead milling. The lysed sample then enters the binder chamber where a solid mixture of guanidine hydrochloride and sodium bisulfate (binder) is dissolved in the sample to facilitate binding of the DNA to the purification filter in the next step.

As a proof of concept for this method, we obtained the lysis efficiency from a sample of Bacillus subtilis spores lysed by a lysis module. Spores represent the most difficult case to lyse. For non-spore applications, the bead milling chamber may be replaced with a chamber module without an electric motor to simplify the design and reduce cost.

And (5) purifying. We face several major challenges in developing a technical approach for microfluidic DNA purification. For example, we need to develop a method to replicate a laboratory process that requires 7 steps and 30minutes as well as a centrifugation step to successfully lyse and purify spore samples. In addition, we needed to elute the DNA in the first 6. mu.L fraction. In laboratory experiments, purified DNA is typically eluted in a larger volume (e.g., 20. mu.L) and then a smaller volume aliquot (1-3. mu.L) is used for PCR amplification. In this case, the DNA was mixed and thus the elution rate was averaged over the entire 20. mu.L. Microfluidic methods involve little mixing because most of the flow is laminar; therefore, the highest concentration of eluted DNA needs to be in the first fraction.

Table 3. we performed three replicates of bacillus subtilis spore lysis using our lysis module. From the following by 10⁷The percentage of the starting mother liquor of individual spores shows the results (based on viability counts). The control was a Claremount bead mill apparatus. Our microfluidic results are comparable to controls using laboratory tips and test tubes.

Bacillus subtilis sample number	Based on 107Viability count input post-lysis recovery (%)
		1	48.4
2	31.6
		3	18.6
Average	32.9
		Control	39.6

On the mfc, after lysis, the sample flows through the purification module, the DNA binds to the filter, and the contents of the remaining lysed sample flow to the waste reservoir. The wash buffer from the reagent cake then flows through a filter to remove residual impurities and is also collected in a waste reservoir. Air is blown through the purification module to dry the filter, followed by elution buffer from the reagent block (the key aspect to achieve significant elution in the first section is elution buffer pH), which, when flowing, removes the purified DNA from the filter in preparation for PCR. Table 4 presents data for three replicates of e.coli samples purified on a microfluidic card and compares these results with those from standard purification filters. In each case, it is clear that the highest concentration is from the first fraction. Each fraction represents DNA in about 6. mu.l of eluate.

Table 4. we repeated 3 replicates of e.coli sample purification using our mfc protocol. We developed a protocol to elute most of the DNA in the first fraction, since using the general laboratory platform method we would not have the opportunity to collect the entire elution and select only one fraction with the tip. The results clearly show that the maximum concentration is from the first 6- μ l fraction.

PCR amplification and labeling. Carrying the purified DNA from the filter, the elution buffer fills the PCR master mix chamber, which contains the lyophilized master mix. The rehydrated master mix then flows into the primer chamber and rehydrates the dried primers. We separated the primers and mastermix for reuse of the universal PCR mastermix module. Lyophilization of oligonucleotides is rapid, and this approach enables us to support rapid development of kits to test emerging threats. After the rehydration step, the sample, along with the master mix and primers, enters the PCR chamber where the sample DNA is then amplified.

Two key features enable us to perform rapid PCR: (1) aluminum PCR module surface and (2) new TEC assembly that can be warmed to >15 ℃/s with an accuracy of ± 1 ℃.

The PCR module was sandwiched between 2 TEC assemblies. The PCR master mix was contained in a 1-mm-high acrylic chamber enclosed by 2 25- μm aluminum walls. The aluminum surface of the PCR chamber enables rapid heat conduction to and from the TEC assembly to the liquid PCR mixture. As a comparison, a 50- μm-thick plastic reduces the temperature-rising plastic by about 3 times.

SRI designs and tests custom TEC assemblies. We have transformed the RMTltd design for fabrication. The custom SRI assembly incorporates a heat sink, a TEC, a feedback sensor (thermistor), and an aluminum nitride (AlN) heat sink around the sensor. The thermistor sensors are calibrated to allow HHA to compensate for any sensor manufacturing error tolerances.

As a proof of our PCR system concept, we generated a double-stranded PCR assay against the aggregated adherent-pilus (AGG) and Shiga toxin (STX2) genes associated with the Escherichia coli O104: H4 pathogen, outbreak in Germany in 2011. Primers and probes for the unique region were selected based on sequence analysis uploaded by BGI immediately after the start of the outbreak. Laboratory tests using bench top equipment were used to establish probe selection and primer optimization for AGG and STX2 target optimization. Once established, the assay was converted to an SRI microfluidic PCR system. Since the SRI PCR system takes advantage of aluminum PCR chambers optimized for thermal conduction and temperature uniformity and new thermistor driven TEC design for rapid temperature cycling, the custom master mix formulation containing adjuvants for passivating the aluminum chambers allows for double-stranded amplification of AGG and STX2 targets within 16 minutes.

The results provide a coverage of 40 PCR cycles (denaturation step at 95 ℃, annealing at 62 ℃ and extension at 73 ℃). Each cycle was carried out for 40 cycles lasting 21 seconds for a total of 14 minutes of PCR thermal cycling. The remaining time was used for initial denaturation and uracil-DNA glycosidase (UNG) treatment. Our custom master mix contains dUTP as part of the amplification and the UNG step ensures that there is no contamination between different uses of the hardware platform. We tested 16-min PCR on 5 different input copy numbers (10, 50, 100, 500, and 1000) and quantified the amplification factors simultaneously for the AGG and STX2 genes. In addition, we tested 500 copy input using a 12-minute PCR protocol and observed that the cycle time decreased from 21 s/cycle to 15 s. Also, with 2 minutes UNG treatment and initial denaturation, the cycle time was only 10 minutes. Table 5 shows the results of nested PCR against amplicon standards to quantify the amplicons in each sample and determine the amplification factors. Overall, our amplification range for modular PCR was 1.6x10⁹To 3.4x10¹¹. In addition, amplicons generated with the modular PCR system have been detected with the SRI modular hybridization system.

TABLE 5 quantification of modular PCR samples. The results show that all samples had >3.4nM amplicon in the hybridization, which is higher than 1-nM LOD.

And (6) detecting. To detect the presence of a particular target, the amplified sample is hybridized to a microarray on an optical waveguide chip. To facilitate hybridization, SSPE buffer from the reagent block is first added to the amplified sample. 2 measurement chambers were used to achieve the optimal ratio of sample to SSPE buffer.

SSPE and PCR are mixed and then pushed into the detection chamber, which incubates the sample on a microarray on an optical waveguide chip. After 5 minutes of temperature-controlled hybridization, using our custom TEC assembly, additional SSPE buffer was flowed into the detection chamber to wash out the sample and remove non-hybridized amplicons, followed by raising the temperature to remove any amplicons that were cross-hybridized or non-specifically bound to the wrong probe.

Finally, the DNA microarray is imaged using custom illumination, collection and imaging optics. The optics block is designed as a separate subassembly that includes everything needed for laser illumination and microarray hybridization imaging. The optical blocks may be prearranged and adjusted prior to installation in the HHA. Once this pre-alignment is done, no further adjustment is required at the time of installation.

The laser irradiation optical element is composed of a line-generating laser diode module and a turning mirror. The target of the laser irradiation is a grating on the microarray chip. Line-generating laser diodes emitted a 10-mW beam at 635nm in a rectangular pattern to excite Alexa Fluor 647nm dye molecules for observation of hybridization to the microarray. The line-generating diode module has focusing and line-generating optics integrated in an off-the-shelf package (bonding limited).

The microarray imaging optics comprise a folded 1:1 relay and an interference filter. Custom designed relay lenses are fast (f/1.5) to maximize light collection capability. It is small in size (12 mm in diameter and <27mm in length). The relay mirror is designed to collimate the light sufficiently into 2 interference filters (made of dielectric stack) that prevent the laser excitation light and scattered light from reaching our monochromatic CCD camera.

The CCD chip and electronics are directly connected to the microarray imaging optics to reduce noise and minimize signal loss. The CCD is capable of sufficient read-out of microarray analysis speed (4 frames/s) and sufficient signal-to-noise ratio for microarray imaging without CCD cooling.

As a proof of concept for the optical system, we hybridized the PCR amplicons generated by the TEC assembly and the SRI aluminum PCR chamber and read the results using the optical system described. The protocol tested included spotted probes (as identified by our rapid DNA synthesis instrument), on-board incubation at 37 ℃ for 5 minutes, multiple stringent washes at temperatures increasing from 37 ℃ to 60 ℃, and optical packaging for automated image capture. We hybridized the negative control with a single control (a3) analyte, the resulting amplicon from 10 copies entered PCR, and the amplicon from 50 copies entered PCR. The test results have been demonstrated by hybridization of a negative control sample and a sample amplified from individual primers for AGG and STX 2. Hybridization images demonstrated that the target probe was clearly visible in both the 10 and 50 input template cases, and absent in the a3 control case.

Low cost mass production of spent mfc. We developed a manufacturing process plan to reduce the cost of fabrication of the mfc for mass production. Because of the simple and inexpensive method of injection molding to create parts, we injection molded the upper and lower layers of the card, as well as the lysis module and reagent block. The remaining laminate layers of the card are manufactured in an automated roll-to-roll process in which rolls of material are laminated together, laser cut, and then rewound onto another roll, all in rapid pipeline fashion on the same equipment system. With high throughput, on the order of millions of cards, the cost can be as low as $ 10/card.

A DNA detection method. Our system has the ability to photolithographically synthesize oligonucleotide arrays in less than 10 hours with all possible probes to amplicons, as we can select the appropriate probes and transfer the platform PCR assay into our platform in about 2 weeks. As shown herein, we have successfully demonstrated this ability to develop amplification assays, convert them to iMFC PCR, and select probes against E.coli O104: H4.

A method for detecting viral RNA. RNA virus detection all the same modules as DNA detection are used with microfluidic assays for reverse transcription and amplification of RNA virus genomes. We have demonstrated the concept of this approach using experiments that can distinguish between seasonal and swine flu. Methods for developing influenza assays are general and the same methods as for selecting agent RNA viruses can be used.

To identify influenza, our first step is to identify primers that selectively amplify a segment of the influenza a virus matrix gene and the portion of the hemagglutinin gene that distinguishes H1 (porcine) and H3 (seasonal) strains. Our RT-PCR protocol includes a 5 minute Reverse Transcription (RT) step at 42 ℃, where the reverse primer anneals to the RNA target and initiates synthesis of the first DNA strand. 2-minute reverse transcriptase inactivation and simultaneous Taq polymer "hot start" is followed by allowing the forward primer to anneal to the first DNA strand and synthesize the second DNA strand. In this regard, 40 cycles of PCR were performed: 10-s denaturation at 95 ℃, 20-s annealing at 62 ℃, and 5-s extension at 75 ℃. Once good amplification was achieved on the bench-top device, we transferred to our modular amplification system, which mimics amplification in our mfc. Due to the rapid temperature rise in this system, the above RT-PCR protocol takes less than 33 minutes. RT-PCR products were first assessed using gel electrophoresis and then on a microarray. Gel electrophoresis results of modular amplification of the matrix and hemagglutinin genes were obtained for CA 2009 swine influenza strain (H1N1) and HK 68 seasonal strain H3N2 (triple amplification). The 244bp amplicon shows the influenza a virus matrix gene. The hemagglutinin amplicon of the H1 strain was 173 base degrees, while the H3 amplicon was 177 base pairs. Hybridization with sequence-specific probes on DNA microarrays gave a clear differentiation of H1 and H3 strains.

The first step in achieving sensitive and selective detection on microarrays is to prepare lithographically synthesized probe selection chips containing 20-25 mer probes to our target of interest that cover the entire amplicon. The ability of us to synthesize all possible probes for the amplicon of interest in less than one day indicates that we can empirically test both sensitive and specific probes. Next, we performed hybridization experiments on the microarray to select the most sensitive and specific probes. A selection of probes that readily distinguished H1 and H3 was identified; there are a few amplicon regions that are readily differentiated between the 2 amplicons. The best probes can be fitted with amine modifications for spotting on epoxy functionalized microscope slides or waveguide chips used in our handheld device.

And (3) an mRNA detection method. Gene expression profiles of peripheral mononuclear cells (PBMCs) are useful methods for monitoring the presymptomatic diagnosis of disease states, environmental exposure and infection. Our mfc capable of mRNA expression analysis uses a slightly modified version of the hardware platform-extra TEC and increased illumination uniformity on the microarray, and can do:

-PBMC selection and lysis: commercially available size exclusion filters for purification of PBMCs from whole blood, and cell selector modules as described herein were used.

-mRNA purification: the microspheres coated with poly-T oligonucleotides were used to capture mRNA from lysed cells.

-T7 linear amplification and labeling: existing PCR chambers were used for T7 amplification and labeling as well as commercially available kits.

-rapid hybridization: determining an analyte concentration using a dynamic measurement of hybridization; the time required for gene expression hybridization is reduced from many hours to several minutes.

The T7 amplification utilized existing iMFC PCR modules and commercially available kits.

And (5) mRNA purification. For mRNA purification, we use a microfluidic purification module that can hybridize mRNA to poly-T coated beads, wash away contaminants, and then release mRNA from the poly-T beads. We used a mixer that kept the approximately 5 μm microsphere beads in solution and used new TEC to melt the mRNA captured from the beads after the washing step. We have tested the device to verify that the 5- μm beads moved well between the bellows mixing chamber and the filter. We have also demonstrated that the fluid can be heated to about 80 ℃ to allow denaturation of poly T: mRNA double strands.

And (4) carrying out rapid hybridization. To reduce hybridization time and improve reproducibility, we derived the concentration from the dynamic rate parameter. In the case of an excess of target transcripts compared to the number of probes, the signal value versus time should follow a dynamic curve:

Y＝C(1-e^-rt),r＝k_off+[A]k_on

in the equation, Y is the background subtracted signal; k is a radical of_offAnd k_onIs a dynamic parameter that depends on temperature, gene sequence, and probe displacement; [ A ]]Is the concentration of the target gene in solution; and C is a slope factor that depends on a variety of factors including light intensity, probe density, target concentration, and dynamic parameters. Our probe selection technique estimates k by correlating the equation with the time series of hybridization signals at various concentrations_offAnd k_on. These estimates can be stored and used in real time to estimate [ A ]]. Because variations in light intensity and probe synthesis density affect parameter C rather than variations within the index, this dynamic technique significantly improves replication repeatability between and on chips.

As proof of concept, we have experimented with the dynamic rate approach, starting with a synthetic 25-base pair oligomer target. Dynamic response of one probe to a3 control labeled oligonucleotide. Each line represents the response for a different concentration a 3: 50nM, 100nM, 300 nM. The parameters C and r for each time series are estimated using nonlinear least squares fitting. Then, experiments at various concentrations [ A ] showed an affine relationship between r and [ A ].

We have identified compounds at r and [ A ]]Conditions that create a sensitive, repeatable relationship therebetween. Our data show that this relationship can occur in the correct direction for [ A ]]nM, (50, 100, 300) r ═ s (0.0031, 0.0033, and 0.0058)^-1. Another benefit of dynamic curve fitting is that the instantaneous equilibrium value C proves to be better than the dynamic rate r, the fitting process is balanced over short-term noise. We note that the ability to measure dynamic parameters depends on the mfc using evanescent waves to excite only fluorescent molecules that bind to the chip surface, giving high signal-to-back ratios even when the target solution is in place.

A method for detecting a protein. One way to expand the detection capabilities of the mfc platform is to include protein biomarkers in microsphere immunoassays that use antibodies labeled with oligonucleotides to generate nonlinear amplifiable DNA targets in the presence of target antigens, and we have developed immunoassay modules with the mfc platform based on SRI-proven microsphere immuno-PCR assays (MSiPCR). Biotinylated target-specific antibodies were conjugated to streptavidin-coated 6- μm polystyrene beads for initial capture of the target antigen. Independent target-specific antibodies are chemically coupled to an oligonucleotide for a secondary capture event of the target protein. Extensive washing between 2 protein capture events washed away any unbound target as well as free conjugate. The remaining bead fraction was then amplified using a specifically labeled primer set and a taqman probe. The amplified nucleic acid signals are then hybridized to a microarray for detection and reading. We have transferred bench-top experiments into the mfc platform: our proof of concept module system of the mfc platform incorporates a bellows mixer for washing and incubation steps, and a filter for capturing beads after nucleic acid amplification.

And (5) improving the system. The optical module can be changed to provide a more uniform pattern in the microarray grating and 532nm excitation is used to improve the detection signal-to-noise ratio. The process of switching from 635nm to 532nm excitation light source is only a matter of changing the grating pitch.

This modified optics block consists of both laser target illumination optics and microarray imaging optics. The laser target illumination optics use a small Diode Pumped Solid State (DPSS) laser module, a scanning focusing optic, and a 2D scanning microelectromechanical system (MEMS) mirror. The DPSS laser module can output a 40-mW laser beam at 532nm for excitation of Cy3 dye molecules. The beam may be focused onto the MEMS mirror using focusing optics. The 2D MEMS mirror can scan a point on a rectangle and aim the beam at the grating. After reflection from the scan mirror, the beam is directed to a scan lens, which collimates the beam before it enters a grating on the microarray chip.

The system scans the same laser spot over the entire grating, and hence over the entire microarray. This eliminates illumination non-uniformities from the line-generating optical element, which can vary by as much as 25% in intensity. The scanning lens also allows us to collimate the beam before it enters the microarray chip grating. We verified with proof of concept verification for the new scan technique approach. Microarray imaging optics are unchanged from the optics block herein, except for filters of different excitation (532nm) and emission (570nm) wavelengths.

Reference to the literature

Niemz, a., t.m.ferguson and d.s.boyle, Point-of-care nucleic acid testing for infectious diseases, Trends Biotechnol,2011.29(5): pages 240-50.

Bissonnette, L. and M.G. Bergeron, Infectious Disease control by Point of Care Personalized medical Molecular diagnostics (Infectious Disease Management through Point-of-Care Personalized medical Molecular diagnostics). Journal of Personalized Medicine 2012.2(4): pages 50-70.

Foudeh, A.M. et al, microfluidics design and technology using Lab-on-a-chips for pathogen detection for point-of-care diagnostics Lab Chip 2012.12(18): pages 3249-66.

Easley, C.J., et al, fully integrated microfluidic genetic analysis system with sample-in-answer-out capability, Proc Natl Acad Sci U S A,2006.103(51): pp. 19272-7.

Xu, G, et al, independent integrated cartridge for real-time PCR and sample preparation for rapid influenza virus diagnosis (A self-contained all-in-one cassette for sample preparation and real-time PCR amplified fluorescence diagnosis). Lab Chip,2010.10(22): page 3103-.

Ferguson, B.S. et al, Genetic analysis of H1N1influenza virus from throat swab samples in microfluidic systems for point-of-care diagnostics (Genetic analysis of H1N1influenza virus from throat swab system for point-of-care diagnostics) J Amchem Soc,2011.133(23): pages 9129-35.

Lam, B, et al, integrate cell lysed, Polymerase chain reaction-free samples into the result bacteria assay within 30minutes (Polymer chain reaction-free, sample-to-answer bacterial detection in30minutes with integrated cell lysis). Anal Chem,2012.84(1): pages 21-5.

Chen, D.et al, Integrated self-contained microfluidic cartridges for isolation, amplification, and detection of nucleic acids Biomed Microdevices,2010.12(4): pages 705-19.

Chen, Z, et al, Development of a general microfluidic device for simultaneous detection of antibodies and nucleic acids in oral fluids (Development of a genetic microfluidic device for detection of antibodies and nucleic acids in oral fluids.) Biomed Res Int,2013.2013: page 543294.

Schumacher, S. et al, high-integrated Lab-on-Chip system for point-of-care multiparameter analysis Lab Chip 2012.12(3): pages 464-73.

Embodiments of the invention include:

1. an automated nucleic acid analysis system comprising sample lysis, amplification, PCR and detection modules in microfluidic communication arranged to perform parallel detection of different nucleic acid sequences by multiple sequence amplification and simultaneous microarray hybridization reading.

2. The system of the preceding or following claim, wherein: the detection module comprises microarray detection optics comprising a microarray scanner employing evanescent wave excitation and detection; the detection module comprises an automated hybridization processor configured to provide a plurality of stringencies by temperature; and/or the PCR module is configured to perform reverse transcription and PCR in a single reaction.

3. The system of the preceding or subsequent claims, comprising an integrated microfluidic card comprising a module and an analyzer, the analyzer comprising a receptacle configured to receive the card, an operator configured to operate the card, and a controller configured to electronically control the operator, the operator comprising a fluidic actuator, a PCR thermal cycler, and an automated hybridization processor and microarray detection optics.

4. The system of the preceding or subsequent claim, further comprising a reagent module configured to contain and deliver reagents to the lysis, purification, PCR, and detection modules.

5. The system of the preceding or following claim which is:

the method is portable: less than 2000, 1000 or 500in³And less than 50, 25, or 10 lbs;

the method is rapid: less than 60, 120, 180, or 240 minutes of analysis;

multiple: analyzing more than 10, 50 and 500 target sequences simultaneously; and/or

Automatically: no user intervention is required between sample introduction and result presentation.

6. The system of the preceding or following claim, wherein:

the sample comprises a protein analyte and the system is further configured to label the protein analyte with a tag comprising a nucleic acid sequence;

the anchored probe defines a sequence by its spatial position;

using fewer primer pairs than the number of sequences analyzed to achieve/effect amplification;

the different nucleic acid sequences are of multiple species/organisms;

the PCR module comprises a metal (e.g., aluminum) PCR reaction chamber;

the microfluidic communication comprises a gas permeable membrane configured for removing gas bubbles, wherein the gas permeable membrane is below the channel layer such that the entire channel is exposed to atmospheric pressure (in a specific embodiment, the membrane spans the card as it is more easily layered than a separate sheet, although it only functions below the channel layer);

the amplification is contained entirely in a consumable (e.g., a non-open tube); and/or

Detection is based on probe sets rather than primer sets (new assays are easily constructed).

7. The system of the preceding or subsequent claim, configured to:

amplification in a single vessel (no sample lysis);

receiving and processing a sample of blood, saliva, GI sample, urine, wound swab, spinal puncture, nasal swab, veterinary and agricultural derived analyte;

receiving a sample via a specimen collection vehicle or transport medium;

treating a sample volume of 1-100 ul;

are modular (modules are interchangeable to support different applications);

can be measured (by channel size and bubble removal); and/or

Are one-way or self-sealing (to prevent cross-contamination of samples).

8. The system of the preceding or subsequent claims, comprising an integrated microfluidic card comprising the module and an analyzer comprising a housing (cartridge) and comprising a receptacle within the housing arranged to receive the card, wherein the analyzer:

the cards were ligated for lysis, purification, PCT (amplification and labeling), and detection;

interaction with the sample by pressure (e.g., sample transport), magnetic field (e.g., sample mixing), temperature (e.g., amplification, stringency, hybridization), and/or light (e.g., hybridization detection); and/or

Detection is performed by coupling an evanescent wave to the sample to observe hybridization in real time and/or to determine possible base pair mismatches and kinetics of generating sequence information.

9. The system of the preceding or subsequent claim, comprising a system of integrated microfluidic cards (cartridges) containing the modules, wherein the cards are arranged to:

specific for the type of disease (e.g., respiratory disease);

specific to the type of patient (e.g., pediatric);

specific for the pathogen type (e.g., biological warfare agents);

specific to the individual (e.g., pharmacogenomics);

a unique identifier containing information specific to the patient;

for single use to maintain sterility and minimize cross-contamination;

produced using a roll-to-roll production step; and/or

From a polycarbonate chassis, a metal foil PCR chamber, an acrylic component, a breathable film material, and/or a polyurethane seal.

10. The system of the previous claim, functionally integrated with a microfluidic fluorescence-activated cell sorter (μ FACS) configured to provide hydrodynamic and/or inertial focusing for particle or cell alignment and comprising microporous electroactive polymer (EAP) actuators configured for sorting.

11. A method comprising parallel detection of different analyte nucleic acid sequences by multiplex sequence amplification and simultaneous microarray hybridization reading using the system of the preceding claims.

Other embodiments and examples thereof are detailed: high efficiency fluorescence activated sorting (FACS) for new point of care, portable and highly integrated applications.

High-end commercial FACS equipment achieves sorting rates of 10,000 to 40,000 sort/s by electrostatically deflecting cells contained within charged droplets [1,2 ]. They can be applied in a wide range of applications, for example, the isolation of cancer-targeting T cells for immunotherapy, the enrichment of stem cells for tissue engineering, and the isolation of specific cells for manipulation or further analysis (e.g., DNA sequencing, RNA expression, and fluorescence in situ hybridization). However, their large and expensive equipment, which requires expert operators and is therefore not suitable for point-of-care or portable applications. In addition, their integrity means that they cannot be easily integrated with other instruments or processes.

In an attempt to address these limitations, researchers have developed many microfluidic FACS variants. Until recently, these devices were slow (more than 1 order of magnitude slower than conventional FACS) and most were difficult to manufacture or unsuitable for manufacturing and practical use. With the development of Pulsed Laser Activated Cell Sorting (PLACS), the state of microfluidics in the art has increased to about 10000 cells/s and has high purity [3 ]. In a PLACS, cells are sorted using a fluid jet created by rapid expansion and contact with plasma bubbles. While fluidic devices are simple, connected, and portable, the system requires a high repetition rate of pulsed laser light, which is expensive and large. Thus, PLACS does not address the challenges of scale and expense.

We disclose a simpler method of EAP-based fluid actuation, which is a polymer that changes shape in response to electrical stimulation. They have been used in microfluidic devices to change the cross-sectional geometry of channels, generate small injections for electrophoretic separation [4], change the fluidic resistance of channels and clear blockages [5 ]. We have developed and integrated new high efficiency microfluidic EAP (μ EAP) actuators into micro-FACS.

The electroactive polymer actuates. Our fluid actuator includes a closed-end fluid chamber in which one or more surfaces comprise an EAP. In one embodiment, the bottom surface of the chamber is an electrode covered with a thin (about 12 μm) layer of dielectric elastomer (silicone) EAP. Conceptually, silicone acts like a flexible capacitor. Which distorts when a voltage is applied to the electrodes, increasing the chamber volume and drawing fluid into the chamber. When the voltage is increased, the silicone relaxes and pushes the fluid away from the chamber.

EAP actuators are easily manufactured using proven small scale manufacturing techniques. To create the actuator, we patterned the electrodes on an indium tin oxide coated slide, which became the substrate. We then screened a layer of the uncured prescription on a slide and allowed it to cure thermally. To create the channel layer, we use conventional soft lithography to mold the microfluidic channels. We then completed the device by aligning and plasma bonding the channel layer to the silicone coated slide. The compatibility of actuators with soft lithography means that they can be easily integrated into large existing libraries of microfluidic devices. It also enables rapid prototyping. Such devices are inexpensive because they only require a voltage source for actuation and the manufacturing method is suitable for low cost manufacturing.

In a specific example, we make<1mm²The actuator of (1), which demonstrates a response time of 10 μ s; however, the actuator dimensions may also be in the order of microns (e.g., less than 1, 10, or 100 μm)²) And the response time can be less than 10, 1 and 0.1 mu s, and shorter response time. Their size options make them particularly suitable for handheld and integrated applications and for parallel operation.

The rapid response rates (<20 μ s) of these exemplary EAP actuators also indicate their sorting rates of >25,000 cells/s. The devices are inexpensive because they require only a voltage source to actuate and are constructed using low cost microfabrication techniques. The use of silicone as a polymer enables simple integration of EAP actuators with microfluidic channels by soft lithography. This enables rapid prototyping and has the potential to scale up to manufacturing levels. EAP μ FACS delivers equivalent flux to desktop FACS in a handheld fashion.

We have optimized the performance of EAP μ FACS and incorporated an improved device into the cell concentration/sorting module integrated with the mfc system. This module is a systematic augmentation of operational encapsulation by concentrating large volumes of dilute cell concentrates (e.g., bacteria present in environmental samples, some bacterial cells per ml) or sorting out selected cells from a background of many cells (e.g., sorting out activated T cells from whole peripheral blood mononuclear cells). Embodiments of the micro-sorter are described further below.

And (4) designing a classifier. The classifier performs 3 key functions: and (5) comparing, detecting and sorting. Our design (e.g., fig. 9) incorporates multiple innovations, including combining hydrodynamic and inertial focusing for alignment, and EAP actuation for rapid sorting. In conventional cell sorters, particles or cells are focused both horizontally and vertically using coaxial sheath flow, which kneads a tight sample stream. Since most microfluidic devices are planar, they can focus particles in only one direction (i.e., horizontally); multilayer devices can also focus particles vertically or longitudinally, but are significantly more complex to manufacture. Without vertical focusing, the particles may be distributed over the height of the channel, resulting in variations in velocity and overlap. In our design, we align horizontally in cross-sectional area by hydrodynamic focusing and vertically in long "neck" by inertial focusing. Inertial focusing occurs when the fluid velocity is high enough to generate lift on the particles [6 ]. This combination enables us to align particles efficiently in a single layer device, which preserves our simple manufacturing process.

For sorting, we first detect particles using fluorescence, where after the target particles are detected, a voltage pulse is applied to one or more EAP actuators. The actuator generates a transient cross flow that deflects the target particle into a new path that channels the sort output, as shown in fig. 9.

FIG. 9 is a schematic of an EAP micro-sorter showing the channel layout of a μ EAP FAC. The inset shows the main function of the channel. Horizontal alignment is achieved by hydrodynamic focusing (left) and vertical alignment is achieved by inertial focusing (middle). Finally, the target particles were sorted by μ EAP actuator (right). The extended contact image shows streaking from unsorted and sorted particles. The flow rate was 8. mu.l/min (107mm/s) and the actuation pulse was 1ms at 400V.

Using a 180 ° asynchronously operating paired actuator significantly improves performance by doubling the force exerted on the fluid and reducing the fluidic resistance, which is proportional to the channel length. With two actuators, one "pulls" and the other "pushes" the fluid, and the fluid moves only a short distance (typically about 2mm) between the actuators. Instead, with a single actuator, fluid movement occurs at the output (about 30mm) from the actuator to the device.

To test our μ FACS device, we used a detection and control system consisting of an epifluorescence microscope, a Charge Coupled Device (CCD) camera, a photomultiplier tube, a field-programmable-gated-array (FPGA) -based data acquisition system, and a voltage amplifier.

Initial particle separation was demonstrated. To demonstrate the separation capability of the μ EAP sorter, we sorted a mixture of green (7 μm) and red (5 μm) fluorescent particles by gating on the green fluorescent signal and applying a 620-V, 500- μ s pulse to the EAP actuator. The fluid flow rate was 10. mu.l/min, which resulted in an average linear velocity of 133 mm/s. Unsorted cells are routed along their default path to the waste channel, while sorted cells are deflected to the path of discharge through the sorting channel (fig. 10). We captured 10- μ l fluid volumes from 2 fluid outputs containing both sortable and unsortable. The volumes were imaged in a separate manual cytometer. Based on the results of the cytometer, we estimated 100% purity and 93% yield.

And optimizing the performance of the actuator. Following our initial proof of concept, we initiated a design study on EAP fluidic actuators to improve our sort throughput. We use COMSOL Multiphysics to develop a simplified actuator electromechanical model. Our results show that most of the actuation occurs at the periphery of the device. Based on these results, we developed a range of actuator designs and tested their performance empirically. By increasing the applied voltage and changing the actuator geometry, we can improve the performance of EAP actuators. We successfully sorted particles with a 25- μ s, 800V pulse at a flow rate of 30 μ l/min (400mm/s), which is a 20-fold improvement over the sorter used in our initial particle separation demonstration.

Cell sorting was demonstrated. To demonstrate cell separation within μ EAP FACS, we prepared mouse lymphocyte samples with fluorescently labeled B cells. Leukocytes were isolated by centrifugation and then fixed prior to labeling with phycoerythrin-conjugated B220 antibody. The samples were input into the FACS. Fig. 11 shows an image of sorted B cells. Sorting was performed with a 100- μ s, 800-V pulse at 11 μ l/min (147 mm/s). Note that the fluorescence breach is brightest in the center. Because the labeled cells are significantly darker than the fluorescent particles, we used a 488-nm diode laser to illuminate the cells in the detection zone, while the darker Light Emitting Diode (LED) lamp provides full field illumination. The bright spots on the right are cells captured on the wall by contaminating fibrils.

Multi-sorter integration. Due to its direct fabrication and compatibility with soft lithography, the μ EAP sorter can be easily integrated into more complex devices. To show the integration capabilities of our EAP actuators, we developed other devices featuring multiple independent sorters. Fig. 12 shows parallel sorting in a two-channel apparatus, with extended time contact showing 2 particles sorted independently in parallel channels, and fig. 13 shows a two-stage series sorting into multiple outputs, with extended time contact showing 2 particle-notes sorted by 2 consecutive sorters into one of 3 bins: bin 3 is the default (unsorted) bin. More advanced multi-channel parallel and multi-stage sequential sorting configurations are similarly constructed.

Our microfluidic fluorescence activated cell sorter (μ FACS) provides all the functions required to sort particles: the particles are aligned, their fluorescent signals detected, sorting decisions made based on fluorescence, and then sorted appropriately. The capabilities demonstrated include: sorting particles with various actuator pulse lengths as short as 20 μ s; sorting with multiple actuator and single actuator configurations; independent parallel sorting in a multi-channel sorter; sequential sorting in a multi-stage sorter. Benefits of microelectric active polymer (μ EAP) sorters include: (a) sorting was triggered rapidly by simple electrical input (certified 20 musec sorting); (b) μ EAP actuators are compatible with a variety of microfabrication techniques; and (c) μ EAP sorter is easy to integrate, parallel and well suited for portable scale devices.

Reference to the literature

[1] Shapiro, Practical Flow Cytometry, John Wiley & Sons,2003.

[2] M.E.Piyasena and S.W.Graves, "intersection of flow cytometry with microfluidics and microfabrication," Labon a Chip,14,1044-1059,2014.

[3] Y. chen, t. -h.wu, y. -c.kung, m.a. teiell and p. -y.chiou, "3D pulsed laser-triggered high-speed microfluidic fluorescence activated cell sorter (3D pulsed laser-triggered high-speed-microfluidic fluorescent-activated cell sorter)," analysis, 138, 7308-.

[4] A.K. price, K.M. Anderson and C.T. Culberson, "Demonstration of integrated electroactive polymer actuators on microfluidic electrophoresis devices," Lab on a Chip,9, 2076-.

[5] C.murray, d.mccoul, e.sollier, t.ruggiero, x.niu, q.pei, d.di Carlo, "electrically-adaptive Microfluidics for active tuning of channel geometry using polymer actuators," Microfluidics and nanofluodiics, 14,345-358,2013.

[6] D.Di Carlo, D.Irimina, R.G.Tompkins and M.Toner, "Continuous inertial focusing, sorting, and separating particles in microchannels," Proceedings of the National Academy of Sciences,104, 18892-.

Embodiments of the invention include:

1. a high efficiency microfluidic electroactive polymer (μ EAP) actuator disposed around the flow channel, wherein a voltage pulse applied to the actuator induces the actuator to create a transient cross flow between the flow channels that deflects target particles within the flow channel to a new road force, wherein the actuator comprises a closed end fluid chamber, wherein a surface of the chamber comprises an electrode covered by an EAP layer of a dielectric elastomer.

2. The plurality of actuators of claim 1 disposed around the flow channel and asynchronous to each other actuators, wherein a voltage pulse applied to the actuators induces the actuators to create a transient cross flow between the flow channel that deflects a target particle within the flow channel onto a new path, wherein each actuator comprises a closed-end fluid chamber, wherein a surface of the chamber comprises an electrode covered by an EAP layer of a dielectric elastomer.

3. A pair of actuators as claimed in claim 1 arranged around a flow channel and 180 ° out of phase with each other, wherein a voltage pulse applied to the actuators induces the actuators to create a transient cross flow between the flow channel which deflects target particles within the flow channel onto a new path, wherein each actuator comprises a closed end fluid chamber, wherein the surface of the chamber comprises electrodes covered by an EAP layer of a dielectric elastomer.

4. The actuator of the preceding or subsequent claims, wherein a plurality of surfaces of the chamber contain electrodes covered by an EAP layer of dielectric elastomer.

5. The actuator of the preceding or subsequent claims, wherein the flow channel is arranged to provide a combination of hydrodynamic focusing for horizontal alignment of the particles and inertial focusing for vertical alignment.

6. The actuator of the preceding or subsequent claim, wherein the new path leads to a sort output.

7. The actuator of preceding or subsequent claims, wherein the fluidic channel comprises a sample input channel and sorted and unsorted output channels, and a new path leading to the sorted output channels.

8. The actuator of the preceding or subsequent claim, wherein the fluid channel is configured for fluorescence detection, wherein after detection of the target particle, a voltage pulse is applied to the μ EAP actuator.

9. The actuator of the preceding or subsequent claims, wherein the EAP layer is 1-50 (or 2-25, or 5-15 μm thick).

10. The actuator of the preceding or subsequent claims, wherein the elastomer is silicone.

11. An actuator as claimed in any preceding or subsequent claim arranged to provide parallel sorting in a multi-channel device.

12. An actuator as claimed in any preceding or subsequent claim arranged to provide multi-stage sequential sorting in a multi-channel device.

13. The actuator of the preceding or subsequent claims, functionally integrated into a fluorescence activated particle sorter.

14. A method of using the actuator of the previous claim, comprising the steps of: the voltage pulses are applied to induce the actuator to create a transient cross flow between the fluid channels that deflects the target particles within the flow channels onto a new path.

The present invention includes all combinations of the specific and preferred embodiments described. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations thereof, are hereby incorporated by reference in their entirety for all purposes.

Claims (11)

1. An automated nucleic acid analysis system comprising sample lysis, amplification, PCR and detection modules in microfluidic communication arranged to perform parallel detection of different nucleic acid sequences by multiple sequence amplification and simultaneous microarray hybridization reading.

2. The system of the preceding or subsequent claim, wherein:

the detection module comprises microarray detection optics comprising a microarray scanner employing evanescent wave excitation.

The detection module comprises an automated hybridization processor configured to provide multiple stringencies by temperature; and/or

The PCR module is configured to perform reverse transcription and PCR in a single reaction.

3. The system of any preceding or subsequent claim, comprising an integrated microfluidic card comprising a module and an analyzer comprising a receptacle configured to receive the card, an operator configured to operate the card, and a controller configured to electronically control the operator, the operator comprising a fluidic actuator, a PCR thermal cycler, and an automated hybridization processor and microarray detection optics.

4. The system of any preceding or subsequent claim, further comprising a reagent module configured to contain and deliver reagents to the lysis, purification, PCR, and detection modules.

5. The system of the preceding or subsequent claim, which is:

the method is portable: less than 1000in³And less than 10 lbs;

the method is rapid: analysis was less than 120 minutes;

multiple: simultaneously analyzing more than 50 target sequences; and/or

Automatically: no user intervention is required between sample introduction and result presentation.

6. The system of the preceding or subsequent claim, wherein:

the sample comprises a protein analyte and the system is further configured to label the protein analyte with a tag comprising said nucleic acid sequence;

the anchored probe defines a sequence by its spatial position;

amplification is achieved using fewer primer pairs than the number of sequences analyzed;

the different nucleic acid sequences are of multiple species/organisms;

the PCR module comprises a metal (e.g., aluminum) PCR reaction chamber;

the microfluidic communication comprises a gas permeable membrane configured for removing gas bubbles, wherein the gas permeable membrane is below the channel layer such that the entire channel is exposed to atmospheric pressure (in a specific embodiment, the membrane spans the card as it is more easily layered than a separate sheet, although it only functions below the channel layer);

the amplification is contained entirely in a consumable (e.g., a non-open tube); and/or

Detection is based on probe sets rather than primer sets (new assays are easily constructed).

7. The system of the preceding or subsequent claim, configured to:

amplification in a single vessel (no sample lysis);

the following analyte samples were received and processed: blood, saliva, GI samples, urine, wound swabs, spinal punctures, nasal swabs, veterinary and agricultural sources;

receiving a sample via a specimen collection vehicle or transport medium;

treating a sample volume of 1-100 ul;

are modular (modules are interchangeable to support different applications);

can be measured (by channel size and bubble removal); and/or

Are one-way or self-sealing (to prevent cross-contamination of samples).

8. The system of the preceding or subsequent claim, comprising an integrated microfluidic card comprising the module and an analyzer comprising a housing (cartridge) and comprising a receptacle within the housing configured to accept the card, wherein the analyzer:

the cards were ligated for lysis, purification, PCT (amplification and labeling), and detection;

interaction with the sample by pressure (e.g., sample transport), magnetic field (e.g., sample mixing), temperature (e.g., amplification, stringency, hybridization), and/or light (e.g., hybridization detection); and/or

Detection is performed by coupling an evanescent wave to the sample to observe hybridization in real time and/or to determine possible base pair mismatches and kinetics of generating sequence information.

9. The system of the preceding or subsequent claim, comprising a system of integrated microfluidic cards (cartridges) containing modules, wherein the cards are configured to:

specific for the type of disease (e.g., respiratory disease);

specific to the type of patient (e.g., pediatric);

specific for the pathogen type (e.g., biological warfare agents);

specific to the individual (e.g., pharmacogenomics);

a unique identifier containing information specific to the patient;

for single use to maintain sterility and minimize cross-contamination;

produced using a roll-to-roll production step; and/or

From a polycarbonate chassis, a metal foil PCR chamber, an acrylic component, a breathable film material, and/or a polyurethane seal.

10. The system of the previous claim, functionally integrated with a microfluidic fluorescence-activated cell sorter (μ FACS) configured to provide inertial focusing and/or hydrodynamics for particle or cell alignment and comprising microporous electroactive polymer (EAP) actuators configured for sorting.

11. A method comprising parallel detection of different analyte nucleic acid sequences by multiplex sequence amplification and simultaneous microarray hybridization reading using the system of the preceding claims.