Patent Application
20150224499
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.
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.
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.
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' knowledge, there are no devices that cover every critical aspect described here. For example, Samsung'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' 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'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.
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.
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.
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.
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.
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.
The specifications of the exemplary device are shown in Table 1 and the detailed configuration is shown in
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.
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
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.
Onsite detection is another main advantage with the designs in
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 (
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
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
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
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
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
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
As an illustration of combining reagent and assay compartments together, one design according to an implementation of the invention is shown in
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.
In the example of
A further simplified design of combination is shown in
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
The design of using a single large-aperture detector for best reliability is shown previously in
A specially designed assay chip loading mechanism is developed as shown in
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.
Because of the open modular system design, this invention could easily accommodate various assay methods, as shown in
All the modules used in the fluidic subsystem (pump, valve, sensor, flow cell, etc.) could be combined with quick connects 40a and 40b. 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.
An example of complete operation procedures are described below:
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.
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 (
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
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 (
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.
A panel of IL6 test with onsite detection was performed with the example system similar to the one shown in
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<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).
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 (<8% variations) with concentrations above 50 pg/mL spiked samples. The recovery became uncertain when the concentration is below 50 pg/mL LOD.
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 (
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
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<150 pg/mL) and higher at high concentration range (average+125% for concentration>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.
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.
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.
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.
| Number | Date | Country | |
|---|---|---|---|
| 61939486 | Feb 2014 | US | |
| 61970684 | Mar 2014 | US |
