Abstract:
An automated assay platform for determining the presence and/or amount of analytes of interest in a sample at point of care integrates microfluidic enhanced assay sites, disposable cartridge designs, a sensitive low-volume detection module, together with selected pumping and valving modules, customized control board and user friendly graphical user interface (GUI). Comparing to traditional assay platform like 96-well ELISA, the platform is capable of reducing reagent consumption, increasing assay speed, and enhancing assay performance with a sample-in-answer-out automated process. This platform also features flexibility of adapting different assay schemes for different analytes.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. provisional patent application No. 61/939,486, filed Feb. 13, 2014, for “Modular Microfluidic Assay Platform and Components”; and U.S. provisional patent application No. 61/970,684, filed Mar. 26, 2014, for “Microassay Devices for Measurement of Biomarkers.” Such applications are incorporated herein by reference in their entirety. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    This invention was made with government support under grant no. W81XWH-09-01-0523 awarded by the Congressionally Directed Medical Research Programs. The government has certain rights in the invention. 
     
    
     BACKGROUND 
       [0003]    This invention relates to assay components, assay devices and methods to improve assay outcomes, and more particularly to the integration of microfluidic technology and detection technology with established assay reagents for automated, fast sample analysis. 
         [0004]    Immunoassay and enzymatic assay technologies for biomarkers are widely used for home, lab and clinical diagnosis. The traditional assay systems with these technologies include microplate based systems, tube/cuvette based systems and strip/lateral flow based systems. Microplate based systems are well established and broadly used in labs and some clinics. This platform still suffers from several drawbacks for point-of-care or home use.
       1. Automation. To make the microplate assay automatic, a considerable amount of instrumentation, such as an automated dispenser, automated plate washer and automated plate changer must be paired with a special plate reader, a capability and resources most small labs and clinics will not have.   2. Portability. Most microplate based assay systems are bulky and not suitable for point-of-care applications. The required movement of optics and microplates would pose limitations on the ability to miniaturize the device.   3. Assay performance. For the most-often-used, 96-well assay platform, there are several assay incubation steps, which require up to eight hours to achieve satisfactory assay performance. The overall average assay time is often longer than four hours. Simply shortening the incubation time will result in much higher limits of detection, often above clinically relevant range of concentrations. More high density microplate platforms (384- and 1536-well) suffer from reproducibility issues and may require an additional robotic system for automated operations, which would significantly increase the instrumentation cost.       
 
         [0008]    Tube/cuvette based systems are broadly used in centralized labs (such as Siemens ADVIA and IMMULITE system, and the Beckman Coulter ACCESS system). They are usually fast and sensitive; however they are not for the territory of point-of-care applications or research use because of the size, cost, special training requirements and availability of assays. Strip and lateral flow based systems are dominating certain biomarker diagnostic fields such as blood glucose and urine hCG level for their simple and fast assay process with very low cost. They are well suited for point of care and home use; however, very few low-abundant biomarkers have attained market success because of more stringent requirements in sensitivity, reliability and reagent requirements, especially for quantification. The most common technique for testing at the point of care (POC) is by use of the so called “Lateral Flow Assay” (LFA) technology. Examples of LFA technology are described in US20060051237A1, U.S. Pat. No. 7,491,551, WO2008122796A1, U.S. Pat. No. 5,710,005, all incorporated in their entirety by reference herein. Another technique for LFA is also described in WO2008049083A2, incorporated in its entirety by reference herein, which employs commonly available paper as a substrate and wherein the flow paths are defined by photolithographic patterning of non-permeable (aqueous) boundaries. Advances in LFA technology are disclosed in applications such as US20060292700A1, incorporated in its entirety by reference herein, wherein a diffusive pad is used to improve the uniformity of conjugation, thereby providing improvements in assay performance. Other disclosures such as WO9113998A1, WO03004160A1, US20060137434A1, all incorporated in their entirety by reference herein, have used the so-called “microfluidic” technology to develop more advanced LFA devices. 
         [0009]    Tremendous efforts have been made to improve the microplate assay performance with a point-of-care platform, among which several instruments based on microfluidic technologies have been developed, such as i-STAT system (Abbott), TROVA system (Siloam Biosciences), and LABGEO analyzer (Samsung) based systems. Microfluidic based systems are ideally suited for assay based reactions as disclosed in U.S. Pat. No. 6,429,025, U.S. Pat. No. 6,620,625 and U.S. Pat. No. 6,881,312; all incorporated in their entirety by reference herein. The key advantages of microfluidic systems are the natural fit for automation, small sample requirement and high surface area to volume ratio that reduces the required assay time. However, it still remains as a challenge to easily adapt more analytes and perform multiple analytes assay simultaneously with a POC platform. It should also be noted that most label-free techniques do not at once meet the sensitivity, specificity, speed and reliability of detection of ultralow levels of many analytes. Immunoassays and enzymatic assays are often needed for specificity and sensitivity, and the protocol involves the use of multiple reagents and washing steps in a sequential programmatic manner—achieving this in the microfluidic format is formidably challenging. Furthermore, the assay results often lack accuracy without internal calibration since most reagents are vulnerable to environmental changes. Many instruments have tried to use a pre-stored calibration curve, but its practical value is limited especially when the assay conditions are changed. The expandability is another important aspect of such a tool, which means that it should be able to adapt new analyte tests or new assay methods easily by adding or exchanging new components. This is extremely helpful in developing new POC assays or performing POC service in resource-constrained environments. To the inventors&#39; knowledge, there are no devices that cover every critical aspect described here. For example, Samsung&#39;s LABGEO analyzer, which is largely similar to the device described in U.S. Patent Application No. 20110269151, covers only several cardio vascular biomarkers without true on-site calibration. The assay format is restricted due to limitations of its centrifugal based fluidic control. Siloam Biosciences&#39; TROVA system, which is based on US Patent Application No. 20120328488, is an open platform that can adapt many assay platforms, but its single channel pipetting fluidic delivery system may introduce cross-contaminations between reagents. Gravity and surface tension controlled flow are susceptible to sample quality and environment changes. Abbott&#39;s i-STAT system, which is related to many patents and patent applications (U.S. Pat. No. 8,017,382, U.S. Pat. No. 8,222,024, U.S. Pat. No. 8,642,322, U.S. Pat. No. 8,679,827, US20030170881, US20090065368, US20110290669, US20130224775), also focuses on several cardio vascular biomarkers besides simple ionic analytes. There is no on chip calibration for these immunoassay based tests so as to allow reliable and accurate quantitation, and they do not accommodate multiple immunoassays to be performed simultaneously. 
       BRIEF SUMMARY 
       [0010]    The present invention addresses limitations of the POC sample analyzer devices described above by introducing a modulated, fully integrated design. Components such as assay cartridges, pumps, valves, detectors, and sensors can be designed such that they are easily exchanged for different assay requirements in different implementations. Among these, several engineering designs and techniques are developed for quick fluidic connections between components, including quick-connect enabled connections, pierce-through self-sealing connections, and compressed O-ring connections. Integrated with specific assay cartridge designs and precise fluidic controls, a sample could in certain implementations be analyzed in less than one hour with built in on-site calibration. Multiple assay methods are easily adapted with different assay cartridges and protocols. Further extended designs are possible for simultaneous detection of multiple analytes. Therefore, the invention disclosed here is applicable not only to enable research lab and clinical diagnostics use, but also appropriate for specifically meeting point of care application requirements and emergency care in various implementations. 
         [0011]    In various implementations, the invention provides a novel automated assay platform for determining the presence and/or amount of analytes of interest in a sample, comprised of uniquely designed component modules and related methods for point of care application. It is a versatile platform with potential of performing any immunoassays and enzymatic assays using a fast, sample-in-answer-out scheme. This platform uses modular designs to integrate disposable assay cartridge, sensitive onsite or offsite detections, precise flow control with pumping and valving system, an effectively error-proof feedback system and user-friendly graphical user interface (GUI). It is specifically designed and constructed to meet the point of care needs that traditional microplate-based systems, biochemical analyzer systems and strip-based systems do not address, because of lack of automation, large sample requirement, poor assay speed, large size of instrumentation and inadequate performance of the assays. 
         [0012]    To perform a test in various implementations, the sample is introduced into the receptacle on the reagent compartment of the assay cartridge. After optional sample pretreatment, the assay cartridge is loaded into the system and the fluidic path is automatically established with the microfluidic system within the chassis with a convenient loading and unloading mechanism by means of quick connects, pierce-through connection, or compress-fitting. The user starts a predefined assay protocol with a user-friendly GUI and the test will automatically be run and the results will be reported once finished. The cartridges are disposable to minimize carryover. With the microfluidic design of the cartridge, the assay time and volume requirement are greatly reduced while keeping the assay performance. Real time calibration is built in with the cartridge so that variations from storage and reagent preparation can be minimized. Simultaneous detection of multiple analytes is also feasible with extended cartridge designs in certain implementations. 
         [0013]    The detailed description and drawings provided herein will offer additional scope to certain implementations of the present invention. It should be understood that the described implementations are provided as examples only. Those skilled in the art will recognize that numerous variations and modifications of the described implementations are within the scope of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0014]      FIG. 1  shows an exploded view of an example system of the present invention wherein all components are integrated with one specific design (top and left sides of the enclosure  10  are not shown for clarity). 
           [0015]      FIG. 2  shows an assay cartridge embodiment with microcapillary assay sites and a quick-connect enabled loading/unloading mechanism. 
           [0016]      FIG. 3  shows an assay cartridge embodiment with inserted microcapillary tubing assay sites and a quick-connect enabled loading/unloading mechanism. 
           [0017]      FIG. 4  shows an assay cartridge embodiment with spiral or serpentine assay sites with an optical quality sealer. 
           [0018]      FIG. 5  shows assay cartridge embodiments with spiral or serpentine assay sites combined for multiple detectors or a single detector with a large aperture onsite detection. 
           [0019]      FIG. 6  shows assay cartridge embodiments with spiral or serpentine assay sites sharing a common inlet or outlet port. 
           [0020]      FIG. 7  shows assay cartridge embodiments with spiral or serpentine assay sites sharing a common inlet or outlet port and features for diffusion limiting. 
           [0021]      FIG. 8  shows assay cartridge embodiments with spiral or serpentine assay sites sharing a common inlet or outlet port and introduced bypassing channel, together with selected optical quality sealer. 
           [0022]      FIG. 9  shows assay cartridge embodiments with spiral or serpentine assay sites integrated with offsite on chip electrochemical detection. 
           [0023]      FIG. 10  shows schemes of assay procedure for internal calibration. 
           [0024]      FIG. 11  shows assay cartridge embodiments with reagent receptacles featuring quick connects for simple fluidic connections with the subsystem. 
           [0025]      FIG. 12  shows assay cartridge embodiments with reagent receptacles featuring a self-sealing pierce-through mechanism for simple fluidic connections with the subsystem. 
           [0026]      FIG. 13  shows assay cartridge embodiments with integrated reagent and assay compartments. 
           [0027]      FIG. 14  shows assay cartridge embodiments with a separate fluidic connection chip. 
           [0028]      FIG. 15  shows the aperture operation for large aperture detectors. 
           [0029]      FIG. 16  shows a chip loading mechanism with compressed O-ring seal. 
           [0030]      FIG. 17  shows an on chip sample preparation with filtration and centrifugation. 
           [0031]      FIG. 18  shows possible assay methods with certain implementations of the invention. 
           [0032]      FIG. 19  is a set of bar graphs showing decreased performance of 96-well plate assay with accelerated steps. 
           [0033]      FIG. 20  is a graph showing system performance with a dye test. 
           [0034]      FIG. 21  is a set of graphs showing system performance with offsite detection. 
           [0035]      FIG. 22  is a graph showing system performance with an IL6 assay. 
           [0036]      FIG. 23  is a set of graphs showing system performance with a T3-T4 competitive assay. 
           [0037]      FIG. 24  shows system performance with simultaneous detection of IL6 and GFAP assay. 
       
    
    
     DETAILED DESCRIPTION 
       [0038]    In various implementations as described herein, the invention features a modular, open design architecture for automated analyte analysis at point of care. A more complete understanding of the apparatus, components and operations can be obtained by reference to the accompanying drawings, as follows. 
         [0039]      FIG. 1  is an overview of an exemplary device. This device has been tested for many protein analytes, especially those related to traumatic brain injury (TBI). It is a fully automated, modular microfluidic platform capable of rapid ultrasensitive analyte detection. It capitalizes on the advantages of using on-chip reaction and detection with sample requirement less than 60 μL, and controlled flow for precisely programmed execution of multistep assay protocol. Other features include:
       1. Low detection limits: demonstrated at 10 μg/mL for IL6 and at 50 μg/mL for GFAP with serum samples.   2. Fast, quantitative results: the disclosed system could simultaneously detect up to four samples with total time less than 1 hour (depending on the specific analyte) for the automated sequence from sample collection to assay results. The use of on-chip calibration enables reliable quantitation due to obviation of chip-to-chip variation.   3. Disposable components: Both reagent cartridges and assay chips are single-use disposable plastic parts, intended to minimize potential cross contamination.   4. Customizable platform: because of the open, modular architecture, the adaptation of new analyte assay is straightforward.   5. On-chip detection: Real time on-chip detection not only increases the detection sensitivity, but also speeds up the assay. It can supply both kinetics and end point information depending on assay requirements.   6. Portable size for POC use: The targeted size of the system is about 9″×11″×15″ with integrated nurse-friendly touch screen.       
 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Specifications of exemplary system 
               
               
                 in FIG. 1 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Assay time 
                 30-60 min 
               
               
                   
                 Sample requirement 
                 60 μL 
               
               
                   
                 Samples used 
                 Serum/plasma/CSF 
               
               
                   
                 Size of assay chip 
                 1.5″ × 1.5″ 
               
               
                   
                 Flow rate 
                 &lt;45 μL/min 
               
               
                   
                 Pressure 
                 0-14 psi 
               
               
                   
                 Detector 
                 PMT 
               
               
                   
                 Dimension 
                 9″ × 11″ × 15″ 
               
               
                   
                 Limit of Detection 
                 10 pg/mL 
               
               
                   
                   
               
             
          
         
       
     
         [0046]    The specifications of the exemplary device are shown in Table 1 and the detailed configuration is shown in  FIG. 1 . This example system is composed of several fixtures and replaceable modules to easily meet different requirements of analyte analysis. The fixtures include the enclosure  10 , chassis  12 , assay chip loading station  14  (assay chip tray  158  and linear actuator  156 ), touchscreen PC  16  and the multifunction control board  18 . The multifunction control board is designed for adopting various modules that could be used in the system, including pumps  20 , valves or manifold  22 , sensors  24 , detectors  26  (both optical and electrochemical), and actuators  28 . Some other useful modules like sample preparation, reagent mixing and light source modules could be also implemented. These replaceable modules make the disclosed system fully open and ready for various analyte analysis. The assay cartridge is fully disposable and a separate design of reagent  30  and assay  32  compartments is shown in  FIG. 1 . These two compartments could be combined to a single embodiment  34  as discussed later. All the components are packed in a light-tight enclosure  10  with at least one opening door  36 , which is used for loading and unloading assay cartridge  34 . More openings are optional, especially for two individual compartment assay cartridge design and for easy maintenance purposes. By using different sets of assay cartridge and a modified assay protocol, different target analytes could be measured in the same way with the same device. For the overall assay process, preloaded assay reagents and samples are loaded from reagents compartments  30  through microfluidic subsystem over to the assay sites  32 . Each step of reagent loading, incubation and removal are precisely controlled by predefined assay programs and through pump  20  and valve  22  systems. The valve options include multichannel valves and manifolds with fluidic control of a plurality of microfluidic connections. The final detection, data analysis and report are processed automatically with the embedded PC system  16 . Several sensors  24  are also integrated for real time assay monitoring and troubleshooting. The sensors include but are not limited to flow sensor, pressure sensor and temperature sensor. An inline flow sensor is very useful to provide real time flow information during the assay and could detect variations caused by clogging, bubbles and valve operations. A pressure sensor that connected to the fluidic system through a manifold could also provide real time flow information to prevent clogging and potential leakage during the assay. A temperature sensor could monitor the local environment for the assay. Once paired with a heating/cooling module, the temperature sensor could help maintain the system operated at the optimal temperature range for assay reactions. Many biomarkers are easily adapted to the analyzer because of its open configurations and some of the tested TBI biomarkers are shown in Table 2. More details of certain biomarkers are described later. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 TBI biomarkers tested with the example device shown in FIG. 1. 
               
             
          
           
               
                 Biomarker 
                 LOD 
                 Dynamic range 
                 Spiked recovery 
               
               
                   
               
             
          
           
               
                 IL6 
                 10 
                 pg/mL 
                 3 Logs 
                 Within 20% 
               
               
                 GFAP-BDP 
                 50 
                 pg/mL 
                   
                 Within 25% 
               
               
                 BDNF 
                 50 
                 pg/mL 
                   
                 NA 
               
               
                 S100b 
                 50-100 
                 pg/mL 
                   
                 NA 
               
               
                 UCHL1 
                 ~0.1 
                 ng/mL 
                   
                 NA 
               
               
                   
               
             
          
         
       
     
         [0047]    A key concept to improve the assay performance with automation herein is the combination of microfluidics with assays. Micro features enable extremely large surface area to volume ratio, so that for diffusion limited assays (including most enzyme-linked immunosorbent assay (ELISA) assays since the kinetics of antibody/antigen reaction is much faster than the diffusion process), the theoretical required assay time and assay volume is greatly reduced (the actual number varies based on specific designs). The automation feature is achieved from the inherent fluidic mode with the interface to precise fluidic control.  FIG. 2  shows an example of assay cartridge design that features microfluidic assay sites  38 , quick-connect enabled fluidic connections  40   a    40   b  and convenient slide-in loading mechanism  42 . Quick-connect enabled fluidic connectors are described, for example, in U.S. Pat. No. 8,337,783 and U.S. patent application Ser. No. 13/417,538, each of which is incorporated by reference herein. The embodiment of the cartridge  32  has six microcolumns  38  across the body with the same diameter (&lt;1 mm). The surface of the microcolumns is modified with analyte assay specific receptors with proper immobilization methods (either adsorption, entrapment, or chemical modification based on surface material, receptors, and coating protocols). All the assay steps are processed on microcolumns and the final signals are measured with a downstream detector. To achieve fast but reliable fluidic connections between the cartridge and the fluidic system in the device, quick connects  40   a    40   b  are used, paired with a sliding station  42 . Quick connects  40   a  are integrated at both ends of the cartridge and they could self-align and connect to the complementary adapter sides  40   b  in the system. One adapter side is fixed while another side is sitting on a moving station  42  so that the engagement and disengagement of the cartridge with the system is freely done and guarantees the full connection force from quick connects. Quick connectors  40   a    40   b  of ¼″ size are demonstrated in  FIG. 2 , but the preferred size could vary depending on number of assay sites and force requirement for reliable connection. The design in  FIG. 2  has to overcome several engineering challenges. The surface area and volume are critical to the assay performance and a reliable manufacturing method is relatively hard to achieve. 
         [0048]    As an alternative design to minimize the potential engineering challenges for the microcolumn features, the whole embodiment  32  in an alternative implementation could be a housing design for embedded capillary columns  44  as shown in  FIG. 3 . Instead of making long microcolumns directly, precoated capillary columns  44  are assembled through much larger apertures  46  on the embodiment  32 . Only the two ends of the embodiment are critical for fluidic connections, which are already handled with quick-connect designs  40   a . Another advantage of this design is that the capillary tubings are available at various sizes with various materials, thus more coating options are feasible for different analytes. Materials such as Polytetrafluoroethylene (PTFE), Polycarbonate (PC), polystyrene (PS), Cyclic olefin copolymer (COO), Poly(methyl methacrylate) (PMMA) and fused silica with ID sizes ranging from 200 μm to 750 μm have been tested successfully with the analyzer. Furthermore, since the surface property of these commercial quality capillary tubings is known, a prescreen process would ensure better microcolumn reliability. It also brings convenience for reliable receptor coating since a batch of long capillary tubings could be coated with the same solution in the same way before cutting into microcolumn sizes. This is a paramount step to improve the overall system performance. 
         [0049]    With the microcolumn design, an example device demonstrated very good performance with model assays as described later, however, faster assays with better performance could not be achieved due to the physical limitations of microcolumns (not compact and no onsite detection). A chip format was therefore chosen.  FIG. 4  shows two design examples of on-chip assay sites that could be used in implementations of the system. Both designs include an embodiment  48  (1-3 mm thickness) with microfluidic channel enabled assay sites  50   a    50   b  and a sealer  52 . Both spiral ( 50   a ) or serpentine ( 50   b ) designs are viable with spiral design providing better flow profile because of less sharp turns on the geometry. The microfluidic features are densely packed for onsite reaction and detection. The channel width could vary from 100 μm to 500 μm. The channel to channel gap could vary from 200 μm to 400 μm and the depth of channel could vary from 50 μm to 300 μm. Smaller overall features may be chosen, but could bring engineering challenges and deteriorate reliability. Each assay site has one outlet  54 , but could have multiple inlets  56  for loading of different reagents as shown in  FIG. 4 . These ports are connected from the back of the embodiment with the sub-fluidic system in the device. 
         [0050]    Onsite detection is another main advantage with the designs in  FIG. 4 , as shown in  FIG. 5 . An optical quality sealer  52  is used to seal the embodiment  48  and optical detectors  58  could align with each assay sites for detections based on either fluorescence or luminescence. Multiple detectors for individual assay sites (such as photodiode array) are an option to minimize the moving parts, but the variations between different detectors could adversely affect the assay results. Instead, single large aperture detector  58  (PMT or camera) with properly designed assay chips could gain the best reliability as an example shown in  FIG. 5 . All the spirals  50   a  are organized within a 1.5″×1.5″ chip  48  so that a large aperture detector  58  (e.g. Hamamatsu PMT H11870-100, CCD camera H10990-904, and Andor CCD camera Luca) could be directly mounted on top of it with an extension tube without additional optics. Both cartridges  48  and detectors  58  are not required to move during the detection and could potentially get the best signal directly from the assay sites. 
         [0051]    Assay chips  48  have ports  54  and  56  open at the backside for fluidic connections. The more open ports, the more complicated a subsequent fluidic connection will be. Thus individual addressable assay sites are expected to have engineering challenges later on for assay automation. Instead, either all inlets or outlets could be combined to one single port  60  to greatly reduce the complexity while keeping a similar or better assay performance ( FIG. 6 ). When used as a common outlet, the potential crosstalk between assay sites is minimal because all diffusions around the common port are easily washed out before entering the assay sites. When used as a common inlet, the requirement on reagents will be minimal since they are not required to route through an external fluidic embodiment (valve, manifold, etc.). The actual optimal configurations depend on targeted assay requirements. 
         [0052]    There are several ways to minimize the potential assay variations due to diffusion from the common port  60  and two of them are shown in  FIG. 7 . One method is to elongate the connection channel from the common port  60  to the assay site  50   a  by introducing another serpentine feature ( 62 ). The longer the serpentine channel, the less effect of potential contamination to the assay site  50   a  from the common port  60 , however, it takes more volume and space on the chip. Thus the configurations are balanced based on the protocol and reagents used. A second approach is to set several fluidic restriction sites  64  on the connection channel ( FIG. 7  bottom with the close-up), wherein narrower and shallower sections  64  could slow down the overall diffusion process while keeping the transition volume even less, with little effect to engineering challenges. The third option for diffusion limiting is to control the surface properties (such as hydrophobicity), however, this might be of limited use because of complexity of reagents used in an assay. Furthermore,  FIG. 7  (top) also shows an example of packing more assay sites  50   a  into the same size assay cartridge, which could be helpful for more simultaneous assays. Actually, there is no theoretical limit to the assay sites per assay cartridge in alternative implementations. 
         [0053]    Since most assays involve multiple reagents, the efficiency of previous solution removal greatly affects the performance of later reagents. Generally, bypassing tubing will be introduced to clean out the solutions in the fluidic subsystem with new solutions and not disturb the assay sites (for previously-described implementations shown in  FIGS. 2 ,  3 ,  6  and  7 ) before passing through the assay sites  50   a  and  50   b . An alternative approach is to include the bypassing feature on the chip itself, as shown in  FIG. 8 . The bypassing channel  66  is a short channel that directly connects the common inlet (outlet)  60  and outlet (inlet)  68  so that a new solution could prime the system without affecting the assay sites  50   a . It is usually designed to be short in length for minimum transition volumes.  FIG. 8  shows a fully functional test example chip  48  with four spiral assay sites  50   a . The chip material could be PC, PMMA, PS, COC or even glass. Opaque material is preferred for on-chip optical detections. The sample chip is 1.5″×1.5″ with 1/16″-⅛″ thickness to keep certain stiffness and prevent from deformation after loading. The channels are 200 μm wide with 140 μm depth. Channel to channel wall is 300 μm thick. Positive control, negative control and a duplicate of samples could be tested simultaneously. Each spiral takes about 3 μL volume and the sample requirement is less than 60 μL. Besides the spiral structure  50   a , some benchmarks  70  are located around four corners for fabrication quality controls. Small wells with different depth and width wells are fabricated together with the spirals to make chip quality control much easier (such as channel depth and wall thickness variations could be checked with these benchmark wells). Three align holes  72  could be used for precise chip mounting, besides an edge reference that could also be used for precise chip alignment with the fluidic subsystem. 
         [0054]    In addition to onsite optical detection with the device, the analyzer can also be adapted to measure assay results electrochemically with offsite electrochemical detectors. Electrochemical (EC) detection as performed here requires the detectable species to be transported (by flow) to the electrochemical sensor. This complication is due to the fact that electrochemical measurements are surface sensitive making it difficult to perform the full assay on the sensor surface. For this reason an example cartridge is shown in  FIG. 9  that permits the assay chip and sensing electrodes to be packaged together. The cartridge is designed in a “layer cake” format with individual layers performing separate functions. As shown in  FIG. 9 , the top layer  74  is the electrical interface layer which contacts the sensor chip  76  through four spring contacts  78  and permits a card edge connector to make electrical contact  80  with measurement electronics. Layer  82  in  FIG. 9  is the plate sealer tape that seals the assay chip  48 . Layer  84  is a double-sided adhesive layer that serves to connect the assay chip  48  with layer  86  which is the fluid interface layer. Layer  86  serves to carry the detection solution from the assay spiral  50   a  in the layer above to the EC detector chip  76  in the layer below. The electrochemical assay cartridge is accessed fluidically through a valve  22  beneath the cartridge. In this case a ten-port selector valve  22  is used to address all the fluid paths of the cartridge. The solution is introduced through the central port  60  and then flows to the respective spiral  50   a  drawn by suction generated by a syringe pump  20 . The fluid then exits the assay chip  48  and passes straight through the fluid interconnect layer  86  and out to the valve  22  for most of the assay steps. During detection, however, the valve  22  switches to pull the solution down a serpentine channel  87  and onto the EC detector chip  76 . After passing over the detector chip  76  the solution again passes though the valve  22  and out to waste. Layer  88  in  FIG. 9  is another piece of double-sided adhesive that not only adheres the fluid interconnect layer  86  to the detection chips  76  but serves as a gasket to form a flow chamber on the detection chip  76  as well. The geometry of this gasket is important to ensure proper flow of reporter molecules from the assay spiral  50   a  across the entirety of the sensing region of the detector chip without permitting the trapping of bubbles. Finally the gasket layer  88  also serves to adhere the fluid interconnect layer to the bottom layer  90 . The bottom layer has recesses  92  that position the detection chips  76  both laterally as well as height-wise so they are able to make proper contact with the adhesive gasket layer  88 . The bottom layer also has four dowel pins  94  that serve to position the layers above. Each layer has a set of four guide holes  96  that align the individual layers. This alignment procedure is enough to enable all the fluid vias to align for the different layers. The fluid vias between the different layers are 500 μm diameter, double the size of the channel widths in the layers themselves to facilitate easier alignment. After assembly the cartridge is placed in a Carver press under 500-1000 psi of pressure to ensure the layers are laminated together properly. 
         [0055]    No matter what detection method is used (onsite or offsite), onsite real-time calibration is another feature that enables reliable assays with the device. Similar to traditional 96-well plate assay, wherein a calibration curve is always prepared together with sample measurement to eliminate uncertainties from reagent degradation, plate differences, concentration variations and environment changes, internal standards are included in this system as shown in  FIG. 10 . A functional reagent compartment  30  includes sample receptacle, standard solution(s), together with other reagents and substrate. They are designed to be physically separated from each other to avoid contaminations during storage. All reagent receptacles are sealed with water impermeable sealers for longer time storage. During a test, all the samples and standard solutions are loaded to different assay sites of the assay compartment  32 , while all other reagents are shared. Reactions on each assay sites  50   a    50   b  could be either parallel or sequential. Parallel reaction means all reagents pass through all assay sites at the same time, which usually requires individual fluidic control for each assay site. On the other hand, reagents pass though assay sites in an orderly sequential mode, which reduces the complexity of the system design. Both options are viable depending on assays. On-chip calibration requirement is also depending on the assay requirements. For triaging assay tests, a cut-off value concentration of standard analyte is enough. It can be used for direct comparison with the measured value and the qualitative test result is a simple yes or no. For more precise quantifications, more assay sites are required to have a full calibration curve on site, similar to that from 96-well plate assay platform. Generally more calibration sites should increase the reliability. A quick test comparing three calibration points and four calibration points with IL6 assay on the system showed more than 10% signal enhancement. The overall number of calibrators has to be determined based on assay performance and system complexity. 
         [0056]    A reagent compartment  30  could be independent from the assay compartment  32  or combined together. Since it contains multiple solutions and interfaces the sample, the loading, and the unloading mechanism is more complicated. There are four main challenges for a user-friendly disposable reagent compartment. First, the reagent should be stored for a long time without leakage/evaporation. Second, loading and unloading the cartridge to the system should be simple. Third, an automatic fluidic connection should be set once loaded. Fourth, it should not have any leakage after unloading of the cartridge. To address all these challenges, two innovative designs are introduced in various implementations of the system. One example design that features quick-connect connectors is shown in  FIG. 11 . The reagent compartment  30  has ten wells  100  to accommodate all the solutions including sample, standards, secondary antibodies, detect reagents, substrate, and washing solution. The bottom of the cartridge features individual quick connects  40   a . Bottom side is sealed with water impermeable sealer (not shown in  FIG. 11 ) while top side is sealed with a cover  102 . When loading, the bottom seal is removed and solutions will remain in the cartridge due to surface tension effect. The compartment  30  could be dropped into the mating adapter  104  on the device. The fluidic connections are automatically established because of the self-alignment feature from the quick connects  40   a  and  40   b . After removing the top cover  102 , the solutions are ready to be pulled into the device. Once the assay is finished, the top cover  102  can be replaced and the cartridge  30  could be safely removed. 
         [0057]    In this quick-connect based design, there is still a chance of solution leakage during cartridge loading and unloading because of risks from capillary force holding solutions. Another design example shown in  FIG. 12  features a pierce-through mechanism. In this design, the fully assembled compartment  30  contains four parts: body  106 , bottom sealer  108 , top cover  110  and top sealer  112 . The bottom sealer  108  is a thick elastic membrane. The top cover  110  is rigid with one large opening  114  for the sample loading and ventilation holes  116  for other wells. Depending on the stability of the substrate, the substrate well might be an empty well or a prefilled one. The prefilled cartridge  106  will then be sealed with low water permeability sealer  112 . The fully assembled cartridge  30  could be stored properly for future use. To load the assay cartridge  30 , the top sealer  112  would be peeled off during the test to reveal the ventilation holes  116  and sample loading well  114 . After loading sample to the sample well  114  with a pipette or similar mechanism, the reagent cartridge  30  would be loaded to the system through a matching adapter  118  with integrated orientation feature. Four magnets  120  are used to keep the cartridge  30  down and secured in place. Unloading of the cartridge is also simplified with this magnet design. There are many needles  122  located at the bottom of the adapter  124  and each of them is aligned with one reagent receptacle. Fluidic connection is established with the needle connectors  122  piercing through the elastomeric seal  108  at the bottom of the cartridge  30 . The material of the bottom sealer  108  is selected for self-sealing of holes after punctured by needles, which is not only important to prevent solutions from leaking during test, but also keep the solutions in place after cartridge removal. The needles  122  are normally protected under a spring  126  loaded guard plate  128  to prevent accidents. Needles  122  are only exposed once the reagent cartridge  30  is loaded to press the guard plate  128  down. After each assay, the operator just needs to seal the cartridge with the original sealer  112  and take it out. The cartridge  30  is ready for disposal without any contamination risks. 
         [0058]    As an illustration of combining reagent and assay compartments together, one design according to an implementation of the invention is shown in  FIG. 13 . Combining the two parts  30  and  32  into one cartridge  34  has several benefits. First, user errors due to mismatched (incompatible) components will be eliminated. Secondly, the error due to misalignment is reduced since it will be virtually impossible to improperly insert the new cartridge with integrated alignment feature. An integrated assay cartridge will also permit better quality control since the reagents and receptor coated assay sites are analyte-specific and will be correlated during manufacture, ensuring lot-to-lot compatibility. This permits the use of a single expiration date for one disposable module. This is important when the system is used to perform multiple analyte measurements. 
         [0059]    In addition to reducing user error, an integrated assay unit permits some simplifications in the device hardware as well. The simplest of these improvements is the use of fewer openings in the device, thus simplifying the light-tight chassis manufacturing. Another improvement enabled by integration is the reduction of transit (dead) volume which translates to shorter assay time and reduced reagent consumption. 
         [0060]    In the example of  FIG. 13 , both reagent  106  and assay  48  components are manufactured by injection molding. This permits small feature sizes and tight tolerances to be preserved on the assay channel molding using the precision mastering. After molding, the assay component  48  has ports  132  drilled and trimmed to size. Then it is coated with receptors and blocked with blocking reagents after sealing with an optical quality sealer  52  on top. The final step is to dry the assay chip  48  before integration with reagent component  106 . The reagent component  106 , on the other hand, has comparatively less stringent molding tolerances. The bottom of the reagent reservoir is sealed with an elastomeric seal  108  that permits access to the reagents by puncturing with needles  122 . There are many vias  130  drilled through the body that match ports  132  from the assay chip  48  to lead solutions. The reagent wells  100  are filled with individual reagents and the assay chip  48  is used to bond on top of the reservoir block  106  by means of the double-sided tape applied to the bottom side of the assay chip (not shown in  FIG. 13 ). The finished cartridges  34  are barcoded and sealed for storage. The use of the integrated assay cartridge is as simple as drop-in the reagent compartment  30  shown in  FIG. 12 . 
         [0061]    A further simplified design of combination is shown in  FIG. 14  for the same chip configurations. The idea is to have all the reagents  134  required for assay (except samples) stored on chip while separated with a septum  136  to cover all the ports  138 . A separate connection channel chip  140  is used to replace the septum  136  and the fluidic connections are automatically established from the reservoirs  134  to the assay sites  50   a  once assembled. The substrate solution could be stored under a separate reservoir  142  with elastomer membrane  144 , which could supply pressure driven flow for all reagents once activated with an actuator. Assay sites  50   a  are spiral configurations similar to other designs as described herein for onsite detection. The overall volume and assay time in this design could be greatly reduced due to extremely small transition volumes. Besides using an actuator, the pumping mechanism could be traditional pumps, or electrochemical pumps for their extreme smooth flow at a flow rate less than 100 μL/min. Suitable electrochemical pumps include those described in U.S. Pat. Nos. 7,718,047 and 8,187,441, each of which is incorporated by reference herein. The complexity of the assay chips will rely on the assay protocol. In the most complicated situation as a full-blown ELISA assay, there will be a total of five solutions and seven steps for one sample measurement. On the contrary, there will be as few as two solutions and two steps with premixing strategy for one sample measurement ( FIG. 14 ). The chip design and the assay performance should be balanced. 
         [0062]    Ideally, one measurement of the sample would be sufficient to give positive or negative answers by comparing to the predefined cutoff value. However, without an internal standard, it would be difficult to correlate the measured signal value with the actual biomarker concentration. Thus a two-spiral chip design is more practical for actual use. As shown in  FIG. 14 , one of the two assay sites  50   a  will be used for sample measurement, while the other site will introduce the biomarker at the cutoff value. By comparing the sample value to that of the “spiked” standard solution, a triaging decision could be quickly made. 
         [0063]    The design of using a single large-aperture detector for best reliability is shown previously in  FIG. 5 . It is possible for a large-aperture camera to define different signals from different assay sites simultaneously. In this case, a complicated image processing method has to be defined in the control software. An alternative way is to introduce a shuttering mechanism, as an example shown in  FIG. 15 . A special designed shutter  146  driven by an actuator  148  is used to expose one assay site  50   a  at a time. It could either be linear actuator  148  as shown, or a rotary shutter as most filter changers do. In  FIG. 15 , the linear shutter  146  is placed close to the chip. One and only one assay site  50   a  is exposed completely once aligned with a predefined aperture  150  on the shutter. The lights from the neighbor sites are minimized with such a close placement and black matte surface around. The measured signal can be directly used for kinetics or end-point analysis without complicated data processing. 
         [0064]    A specially designed assay chip loading mechanism is developed as shown in  FIG. 16  since quick-connect design does not fit because of the geometry constraints and the challenges to seal multiple sites on the same plane in some circumstances. Instead, a spring-loaded actuator  152  paired with compressed O-ring seals for O-ring sealed ports  154  is employed. The key features are the linear actuator  156  and the redesigned assay chip tray  158 . The assay chip tray  158  has a chip insertion slot  160  and three edges are designed to precisely define the position of the chip for fluid connections. In the center of the tray are six raised O-ring sealed ports  154 , which match the ports on the inserted chip  32 . The raised bed  162  feature ensures proper contact between the chip  48  and the O-ring  154 , but not other parts of the assay chip tray  158 , which concentrates the force over the O-rings for better sealing. The linear actuator  156  will raise the assay chip tray  158  together with the chip  32  and against the top optical assembly. It is spring loaded to tolerate certain variations from chip thickness. With properly adjusted actuation force, which could be fine adjusted with an integrated pressure sensor  166 , the fluidic connection between the chip  32  and the valves  22  downstream is automatically set without leakage or clogging and the assay could be started. Just toggling the linear actuator  156  to lower the chip tray  158  and the chip  32  could be removed from the front. Further design could introduce a motorized actuator controlled by the central board  18  with the feedback from the pressure sensor  166  for automatic pressure control. 
         [0065]    Samples that could use our sample analyzer are typically serum, plasma, urine, and CSF. It is possible to use whole blood as a sample with on-site sample preparation.  FIG. 17  shows two examples of onsite plasma preparation with filtration and centrifugation. In Design  1  ( FIG. 17  top), a whole blood reception well  168  is introduced on the reagent compartment and multiple layer filters  170  are fixed between whole blood receptacle  168  and the plasma well  172 . A plunge-type cap  174  is to seal the whole blood receptacle  168 , while pushing blood through filters  170  to a plasma well  172 . The plasma well  172  is connected to the fluidic subsystem and the collected solution is used for sample test. The multi-layer filter membrane  170  is sandwiched between two plates. A star channel feature  176  is located at the plasma side of the filter to collect filtered solution, also to supply the support of filter  170 . Double-side tapes  178  could be used to form water-proof sealing between layers of membranes and between plate  180  and the membrane. Cell lysis could be controlled with the applied force, which is controlled by the depth of inserted plunge  174  and proper area. The efficiency of plasma collection could reach 25% of whole blood in this design. In Design  2  ( FIG. 17 , bottom), a cell collection chamber  182  is introduced on the cartridge  106 , which is connected to the whole blood receptacle  168  with a narrow gap feature  184 . Both wells are located on the line of a centrifugation radius. A cartridge adapter connected to a motor head is used to rotate the whole cartridge  30  and the cell pellets would accumulate in the outside well after centrifugation. Plasma left in the inside well is loaded to the fluidic subsystem after loading the cartridge  30  onto the system through the bottom hole. The efficiency of plasma could reach 50% of the whole blood with proper well designs. 
         [0066]    Because of the open modular system design, this invention could easily accommodate various assay methods, as shown in  FIG. 18 . In theory, any assays that can be captured on site for quantifications could be good candidates, which includes all the sandwich ELISA format with variations ( FIG. 18-1 ), direct ELISA ( FIG. 18-2 ), competitive ELISA ( FIGS. 18-3  to - 5 ) and their variations, and direct enzymatic measurements ( FIG. 18-6 ). It is worth noting that it is possible to mix all reaction reagents together and be captured on site with a different binding mechanism, either Ab-Ag or Avidin-Biotin mechanism, which should greatly reduce the assay steps and time involved, thus leading to a much simplified device design. 
         [0067]    All the modules used in the fluidic subsystem (pump, valve, sensor, flow cell, etc.) could be combined with quick connects  40   a  and  40   b . It is great for prototype development because of its simplicity to switch different modules. Even for the final version of the device, quick connect-based modular design is a good option for cartridge loading and waste container connection. 
         [0068]    An example of complete operation procedures are described below:
       1. System preparation, including system validation and priming. A separate priming protocol may be used.   2. Getting the sample(s) and the appropriate assay cartridge(s)  34 .   3. Optional sample pretreatment with onsite preparation, according to the protocol.   4. Inserting the assay cartridge  34  into the analyzer through the opening cover, or inserting the reagent  30  and assay  32  compartments separately to the specified locations according to the protocol. Either a drop-in or insert-in mechanisms are used for automatic fluidic connections.   5. Identifying, registering and processing information about the assay cartridge  34  into the sample analyzer by means of a user interface  16 .   6. Initiating analysis by inputting a command into a controller  18  located within the analyzer by means of the user interface  16 .   7. Monitoring the real time information displayed on the GUI  16  about the status of the assay such as temperature, flow rate, and the potential error messages.   8. Collecting, analyzing, reporting and storing the analytical data by means of the user interface  16 .   9. Discarding used cartridges and waste per safety rules and replace with dummy cartridges for idle operations.   10. Troubleshooting according to the on-screen display or the manual.   11. Operating maintenance protocol for normal day-to-day operations and dormant protocol for long term storage.       
 
         [0080]    As described before, though 96-well plate assay platform is well accepted as the gold standard for most assays, its performance deteriorates when using an expedited protocol.  FIG. 19  shows the decreased performance of 96-well plate assay with accelerated steps according to one test. A standard sandwich ELISA for the detection of GFAP break down product (GFAP BDP) was conducted with total detection time of about 3 hr 45 min. In this test, monoclonal anti-GFAP antibody (Mab) was used as the primary antibody that was immobilized on the ELISA plate. After blocking with blocking buffer, the GFAP sample was delivered into the plate for incubation; then, horseradish peroxidase (HRP) conjugated polyclonal anti-GFAP antibody (Pab-HRP) was introduced for incubation; finally TMB substrate was added for incubation and the HRP enzymatic product was determined by measuring the absorbance on the plate reader.  FIG. 19  (top) shows sandwich ELISA for GFAP test with limit of detection (LOD) about 250 pg/ml for 4 hr detection time. Following this, 55 min sandwich ELISA ( FIG. 19  bottom) was performed for the detection of GFAP with LOD of about 10 ng/ml. Experimental conditions are shown in the  FIG. 19 . In this example, GFAP assay with different protocols showed that assays with a 55-minute protocol significantly increases the LOD (by more than an order of magnitude) compared to the normally recommended 4-hour protocol. So expediting the protocol by cutting down the time or number of steps simply deteriorates the sensitivity. As shown later in a comparative example, it is easy for the analyzer of the present invention according to certain implementations to obtain better LOD within 60 minutes. 
       Test Example 1 
       [0081]    To check the non-assay related system reliability (including flow, detector, quick-connect components, electronics and software), blue dextran solutions with concentration from 0.0156 to 1 mg/mL were injected into a blank cartridge ( FIG. 3 ) and the signals were measured offsite through a microflow cell with absorbance measurement. The test results from ten repeated experiments are shown in Table 3 and graphically in  FIG. 20 . A linear standard curve is plotted with relative standard deviation (RSD) value below 5% for concentrations over 0.0313 mg/mL dextran, which covers more than 97% of the tested dynamic range zone. For lower concentrations, the reliability is more affected by the capability of the detector and the flow variations. Considering the signals were obtained with moving solutions, the actual variations for assay sites are expected to be even less, which indicates a better detection reliability for on chip detection. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 System reliability test with ten repeated blue 
               
               
                 dextran dye test. 
               
             
          
           
               
                   
                 Cone (mg/mL) 
                 Average signal 
                 Stdev 
                 RSD (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 0 
                 2.49 
                 1.72 
                 68.9 
               
               
                   
                 0.0156 
                 35.55 
                 6.54 
                 18.4 
               
               
                   
                 0.0313 
                 72.18 
                 2.03 
                 2.8 
               
               
                   
                 0.0625 
                 125.13 
                 4.80 
                 3.8 
               
               
                   
                 0.125 
                 168.30 
                 5.66 
                 3.4 
               
               
                   
                 0.25 
                 456.13 
                 5.78 
                 1.3 
               
               
                   
                 0.5 
                 1063.53 
                 24.25 
                 2.3 
               
               
                   
                 1 
                 1875.15 
                 81.91 
                 4.4 
               
               
                   
                   
               
             
          
         
       
     
       Test Example 2 
     Assays with Offsite Detection 
       [0082]    IL6 test with spiked human serum with offsite detection was performed on a test instrument. PMMA capillary tubing coated with mouse anti-IL6 antibody was blocked with blocking buffer and dried for storage. Capillary columns were cut into 10 cm long segments and assembled with the cartridge housing as shown in  FIG. 3 . Reagent compartments ( FIG. 11 ) were prefilled with standard solutions, washing buffers, secondary antibodies, streptavidin-HRP solution and substrate. After inserting both compartments into the system and loading the sample to the receptacle, the assay was performed with a preconfigured program automatically. A typical offsite real time detection signal with IL6 concentration from 0 to 800 pg/mL is shown in  FIG. 21  top. After automatic baseline correction, peak heights at specific timing were measured and the sample concentration could be determined by comparing to the standard solutions. The overall assay time was about 75 min (30 min sample incubation, 15 min secondary antibody incubation, 10 min Streptavidin-HRP incubation and 10 min color development plus washing time), which is much faster than a comparable 96-well ELISA assay (4-6 hours). 
         [0083]    Besides the real time calibrators, a predefined master calibration curve could also be combined with an on chip calibrator to further minimize assay variations. As shown in Table 4, the spike recovery test results of IL6 assays at different concentrations were calibrated with a predefined calibration curve ( FIG. 21  bottom), which was generated based on three repeated assays. The assay procedure was similar to that described earlier, except the final sample data was calibrated against a real-time calibrator adjusted master standard curve. Except the low concentration range, the spike recovery of IL6 assays are all within 10% variations, which is comparable to traditional 96-well plate detection. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Spike recovery test of IL6 samples in human sera. 
               
             
          
           
               
                 Spiked conc. (pg/mL) 
                 Measured conc. (pg/mL) 
                 Recovery (%) 
               
               
                   
               
             
          
           
               
                 100 
                 68.6 
                 69 
               
               
                 200 
                 182.4 
                 91 
               
               
                 400 
                 418.3 
                 105 
               
               
                 200 
                 184.2 
                 92 
               
               
                 600 
                 631.6 
                 105 
               
               
                   
               
             
          
         
       
     
         [0084]    A panel of IL6 experiments with a total of 15 tests over four days is shown in Table 5. In details, PMMA columns with 500 μm ID were coated with priming antibody and cut into 10 cm lengths. Each reagent cartridge contains five capillary columns. One column is used for sample test and the other four are used for real-time calibration for best measurement accuracy. Three to four tests were performed each day with an 88-min protocol. A system cleaning step was used between assays and fresh cartridges were used for all the tests. The internal standard concentrations are 0, 50, 200 and 800 pg/mL IL6 spiked human sera samples were prepared with human serum with concentration range from 50 pg/mL to 400 pg/mL. The results of the panel of experiments showed that the recovery rates are within 32% of variations, while 14 out of 15 tests are less than 25%. Meanwhile, the precision of the system at different concentrations can also be obtained from this panel of experiments and summarized in Table 5. The overall spike-recovery precision is between 82% to 103% with a less than 20% variation. These results already match most commercial 96-well ELISA platforms with serum/plasma tests, obtained with a smaller footprint, much shorter assay time, and with a fully automated process. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Panel of IL6 assay tests with spiked 
               
               
                 human sera. Total of 15 tests in four days. 
               
             
          
           
               
                   
                   
                 Spiked 
                 Calculated 
                   
               
               
                   
                   
                 Concentration 
                 Concentration 
               
               
                   
                   
                 (pg/mL) 
                 (pg/mL) 
                 recovery 
               
               
                   
                   
               
             
          
           
               
                   
                 Test 1 
                 100 
                 78.85 
                 0.79 
               
               
                   
                 Test 2 
                 100 
                 100.31 
                 1.00 
               
               
                   
                 Test 3 
                 100 
                 67.97 
                 0.68 
               
               
                   
                 Test 4 
                 217.9 
                 183.01 
                 0.84 
               
               
                   
                 Test 5 
                 217.9 
                 182.19 
                 0.84 
               
               
                   
                 Test 6 
                 217.9 
                 181.49 
                 0.83 
               
               
                   
                 Test 7 
                 217.9 
                 212.88 
                 0.98 
               
               
                   
                 Test 8 
                 75 
                 64.94 
                 0.87 
               
               
                   
                 Test 9 
                 75 
                 56.43 
                 0.75 
               
               
                   
                 Test 10 
                 75 
                 63.18 
                 0.84 
               
               
                   
                 Test 11 
                 50 
                 57.93 
                 1.16 
               
               
                   
                 Test 12 
                 400 
                 451.69 
                 1.13 
               
               
                   
                 Test 13 
                 400 
                 498.51 
                 1.25 
               
               
                   
                 Test 14 
                 400 
                 374.33 
                 0.94 
               
               
                   
                 Test 15 
                 400 
                 321.12 
                 0.80 
               
               
                   
                   
               
             
          
         
       
     
       Test Example 3 
     Assays with Onsite Detection 
       [0085]    A panel of IL6 test with onsite detection was performed with the example system similar to the one shown in  FIG. 1 . In details, 1.5 mm thick polystyrene assay chips were manufactured with hot embossing. The chips were batch processed for sealing and antibody coating. The final chips were stored dry in the refrigerator for the panel of experiments. The reagent cartridges were machined in house. All solutions except samples were prefilled in the cartridges and stored in the refrigerator before tests every day. Samples were prepared every day with human sera. After sample loading and cartridge/chip assembly, a 67 min protocol (including priming) was used for all the tests. A washing cycle with dummy chip and washing cartridge was performed between tests. Results of 7 days of 28 sample tests are summarized in Table 6. The results show consistent performance between 6.25 pg/mL and 200 pg/mL. The imprecision is less than 25% for most assay conditions. The variation is higher at 6.25 pg/mL, which is below the claimed 10 pg/mL LOD. The corresponding Receiver Operating Characteristics (ROC) curve with a 30 pg/mL cutoff value is plotted in  FIG. 22 . It demonstrated 100% sensitivity and 83% specificity. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Spike recovery test of 28 IL6 sera 
               
               
                 with onsite detection. 
               
             
          
           
               
                   
                 Concentration 
                 Measured 
                   
                   
               
               
                   
                 (pg/mL) 
                 (pg/mL) 
                 stdev 
                 RSD 
               
               
                   
                   
               
             
          
           
               
                   
                 6.25 
                 4.5 
                 2.4 
                 54.5% 
               
               
                   
                 12.5 
                 12.1 
                 2.1 
                 17.7% 
               
               
                   
                 25 
                 22.1 
                 6.1 
                 27.7% 
               
               
                   
                 37.5 
                 37.7 
                 9 
                 24.0% 
               
               
                   
                 100 
                 105.9 
                 16 
                 15.1% 
               
               
                   
                 150 
                 133.9 
                 14 
                 10.5% 
               
               
                   
                 200 
                 174.5 
                 NA 
                 NA 
               
               
                   
                   
               
             
          
         
       
     
         [0086]    Another example of analyte is TBI biomarker GFAP. A panel of GFAP tests in spiked human sera was also performed. In details, 2.2 mm thick white polystyrene chips were manufactured with hot embossing. The chips were sealed and coated with primary antibodies in-house. The final chips were stored dry in the refrigerator for the panel of experiments. The reagent cartridges were machined in-house. All solutions except samples were prefilled in the cartridges and stored in the refrigerator before tests. A modified 67 min protocol (including priming) was used for all the tests. All measurements were finished automatically with integrated detector and control software. A washing cycle with dummy chip and washing cartridge was performed between tests. The intra-assay precision of GFAP test was examined by measuring the same concentration on the same chip at five different concentration levels. As shown in Table 7, the test of GFAP spiked serum samples showed intra-assay CV&lt;15% and the LOD is about 50 pg/mL. Though these results compare well or better to other systems (such as standard 96-well assay (FIG.  19 )), we expect them to be further improved with better quality assay chips (e.g. injection molded chips). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Intra assay CV of spiked GFAP test in human sera. 
               
             
          
           
               
                   
                 Conc. (pg/mL) 
                 Signal (au) 
                 Stdev 
                 CV (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 0 
                 82719.6 
                 9202.8 
                 11.1 
               
               
                   
                 50 
                 141402.2 
                 12093.0 
                 8.6 
               
               
                   
                 100 
                 227471.0 
                 21363.9 
                 9.4 
               
               
                   
                 400 
                 481911.1 
                 59686.8 
                 12.4 
               
               
                   
                 800 
                 747024.9 
                 101731.4 
                 13.6 
               
               
                   
                   
               
             
          
         
       
     
         [0087]    The overall GFAP assay performance with onsite detection system was assessed with a series of spike recovery tests. With similar assay setup and protocol, human sera were spiked with certain levels of recombinant GFAP and 15 test results were obtained in straight 4-day tests. The measured values were compared with the expected amount of GFAP spiked (Table 8). This device demonstrated very good recovery (&lt;8% variations) with concentrations above 50 pg/mL spiked samples. The recovery became uncertain when the concentration is below 50 pg/mL LOD. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Spike recovery test of 15 GFAP sera. 
               
             
          
           
               
                 Spiked Conc. (pg/mL) 
                 Measured Conc. (pg/mL) 
                 Recovery (%) 
               
               
                   
               
             
          
           
               
                 25 
                 13.3 
                 53.2 
               
               
                 50 
                 49.4 
                 98.8 
               
               
                 75 
                 75.8 
                 101.1 
               
               
                 100 
                 107.8 
                 107.8 
               
               
                 150 
                 149.4 
                 99.6 
               
               
                 300 
                 320.9 
                 107.0 
               
               
                 600 
                 591.1 
                 98.5 
               
               
                   
               
             
          
         
       
     
       Test Example 4 
     Adaptation of Competitive Assays 
       [0088]      FIG. 23  shows the system performance of competitive immunoassays for T3 and T4 measurements. In details, the assay sites were coated with streptavidin as the capture reagents. Standard/sample solutions were mixed with specific concentrations of HRP labeled T3 (T4) and biotinylated T3 (or T4) antibodies. The mixture was loaded to the assay sites with the device and incubated for 13 min before washing with washing buffer twice. Signals were measured on site after loading substrate through the assay sites immediately. This assay method is actually similar to that in  FIG. 18-3  except an additional capturing layer was introduced. The competition happened between sample/standard T3 (T4) and HRP labeled T3 (T4) for the binding of biotinylated antibody, which was captured eventually at the assay site. Washing step removes the unbound enzyme conjugates and the final signal is reversely correlated to the concentrations of sample and standards. The data shown in  FIG. 23  is comparable with results from commercial assay kits. It confirms that this invention is capable of measuring analytes with competitive assay methods, which also greatly reduces the assay time required (&lt;20 min). 
       Test Example 5 
     Simultaneous Detection of Multiple Biomarkers 
       [0089]    As a platform system, multiple biomarkers have been proved working on the system. Multiple biomarker detection could be achieved with sequential tests by changing assay reagents/chip with one single instrument. However, the total assay time will be multiplied by the number of biomarkers tested. This is not practical unless more instruments are used simultaneously. To solve this dilemma, an eight spiral assay chip was designed and fabricated for simultaneous dual biomarker detection ( FIG. 24 ). An IL6/GFAP dual assay was demonstrated with spiked human sera. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Sample preparation for dual biomarker test. 
               
             
          
           
               
                   
                 GFAP (pg/mL) 
                 IL6 (pg/mL) 
               
               
                   
                   
               
             
          
           
               
                   
                 Sample 1 (S1) 
                 0 
                 200 
               
               
                   
                 Sample 2 (S2) 
                 50 
                 50 
               
               
                   
                 Sample 3 (S3) 
                 200 
                 12.5 
               
               
                   
                 Sample 4 (S4) 
                 800 
                 0 
               
               
                   
                   
               
             
          
         
       
     
         [0090]    The overall chip dimensions and spiral characteristics remain the same with the eight spiral chips. Two preliminary tests for simultaneous detection of GFAP and IL6 in co-spiked serum samples had been conducted. Four spirals shown in  FIG. 24  were used for GFAP (solid black) measurements and the other four spirals were used for IL6 (broken line) measurements. These spirals were carefully coated with their primary antibodies. To minimize the number of reagents, the standard solutions/samples were prepared as mixtures in a reverse concentration order in human sera (cf. Table 9); thus any potential crosstalk of two assay reagents should reveal if present. The secondary antibodies were also a mixture of the two to save number of reagents used. Thus no modification to the reagent cartridge was required. The protocols were modified for eight-spiral chip tests, but the overall assay time was kept the same (67 min including priming time). The total sample requirement is about 100 μL each, which is about doubled compared to single biomarker test. The results are shown in Table 10. Both GFAP and IL6 measurements were comparable to those obtained with four spiral chips on single biomarker measurements. These results clearly demonstrated multi-biomarker simultaneous detection capability of this implementation of the invention. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Assay results of simultaneous IL6 and 
               
               
                 GFAP measurements with eight-spiral chips. 
               
             
          
           
               
                   
                 GFAP 
                   
                   
                   
               
               
                   
                 (pg/mL) 
                 Signal 
                 IL6 (pg/mL) 
                 Signal 
               
               
                   
                   
               
             
          
           
               
                   
                 800 
                 8223 
                 200 
                 16429 
               
               
                   
                 200 
                 7469 
                 50 
                 4034 
               
               
                   
                 50 
                 5528 
                 12.5 
                 2875 
               
               
                   
                 0 
                 5141 
                 0 
                 1987 
               
               
                   
                   
               
             
          
         
       
     
       Test Example 6 
     Enhancement with Recursive Sample Loading 
       [0091]    Another way to further increase the sensitivity is to use a recursive sample loading strategy. To evaluate this approach, the GFAP assay was performed with all conditions similar to that described earlier, except a modification to the protocol so that the samples would be loaded after all standard solutions. Instead of loading 40 μL sample the same way as the standard solutions at once through the assay spiral, 55 μL samples were loaded four times at one min interval. The overall assay time increased 2 min more to 69 min. The preliminary test result with this approach is summarized in Table 11. Fifteen GFAP spiked human serum samples were tested in 4 days. An enhancement effect from the recursive sample loading was observed compared to previous single loading method. It is lower than the expected value (300% based on four times loading vs one time loading) and lower at low concentration range (average+68% for concentration&lt;150 pg/mL) and higher at high concentration range (average+125% for concentration&gt;150 pg/mL). An adjustment method could be established with more tests to correlate the measured value to the true value, which could further improve the LOD of the system. The potential drawbacks of this approach are longer assay time (because of more sample loading and incubation time) and additional sample volume requirement for extremely low concentration samples. The difficulty is to establish a reliable correlation between the actual and the measured sample concentration after multiple loadings. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Enhancement effect of recursive 
               
               
                 sample loading strategy. 4 days of 15 GFAP 
               
               
                 spiked serum samples were tested. 
               
             
          
           
               
                   
                   
                 Measured 
                   
                   
               
               
                   
                 Spiked conc 
                 Concentration 
               
               
                   
                 (pg/mL) 
                 (pg/mL) 
                 Ratio 
                 Enhancement 
               
               
                   
                   
               
             
          
           
               
                   
                 0 
                 0 
                 NA 
                 NA 
               
               
                   
                 0 
                 0 
                 NA 
                 NA 
               
               
                   
                 25 
                 42 
                 1.68 
                 68.00% 
               
               
                   
                 50 
                 83 
                 1.66 
                 66.00% 
               
               
                   
                 75 
                 117 
                 1.56 
                 56.00% 
               
               
                   
                 75 
                 109 
                 1.45 
                 45.33% 
               
               
                   
                 75 
                 156 
                 2.08 
                 108.00% 
               
               
                   
                 100 
                 181 
                 1.81 
                 81.00% 
               
               
                   
                 150 
                 214 
                 1.43 
                 42.67% 
               
               
                   
                 150 
                 288 
                 1.92 
                 92.00% 
               
               
                   
                 300 
                 730 
                 2.43 
                 143.33% 
               
               
                   
                 300 
                 613 
                 2.04 
                 104.33% 
               
               
                   
                 300 
                 818 
                 2.73 
                 172.67% 
               
               
                   
                 300 
                 727 
                 2.42 
                 142.33% 
               
               
                   
                 600 
                 1330 
                 2.22 
                 121.67% 
               
               
                   
                   
               
             
          
         
       
     
         [0092]    The present invention has been described with reference to the foregoing specific implementations. These implementations are intended to be exemplary only, and not limiting to the full scope of the present invention. Many variations and modifications are possible in view of the above teachings. The invention is limited only as set forth in the appended claims. All references cited herein are hereby incorporated by reference to the extent not inconsistent with the disclosure herein. Unless explicitly stated otherwise, flows depicted herein do not require the particular order shown, or sequential order, to achieve desirable results. In addition, other steps may be provided, or steps may be eliminated, from the described flows, and other components may be added to, or removed from, the described systems. Accordingly, other implementations are within the scope of the following claims. Any disclosure of a range is intended to include a disclosure of all ranges within that range and all individual values within that range.