Sample preparation and analysis presents many logistical problems. Conventionally, many medical samples (such as blood, saliva, urine and swab eluate) are provided to a doctor, for example a general practitioner doctor (GP) or a principle care physician (PCP), in a local surgery without the equipment necessary to analyse the sample. Hence, the sample must be sent to a laboratory where the sample is analysed. The test results must then be collated and returned to the GP to analyse the results and make a diagnosis. This approach is inadequate. Firstly, there is a significant risk that a sample is lost in transit or mismatched with the wrong patient. Moreover, whilst recent developments in technology have reduced the overall time taken to conduct the test, the delay involved in sending the sample to a laboratory is unsatisfactory.
Nevertheless, analytical systems of the kind found in laboratories are complex and it is often difficult to provide sufficient amounts of pure targets from source samples to reliably perform downstream analytical assays. This typically prohibits local GP surgeries from being able to carry out such tests on site.
However, in recent years efforts have been made to reduce the scale of the analytical systems to make tests faster and simpler to run, and require smaller quantities of sample. For instance, “laboratory on a chip” (LOC) devices (a subset of microfluidic devices) integrate almost all medical tests or diagnostic operations performed in a hospital on a single microfluidic chip. The channels forming such microfluidics devices handle small fluid volumes and are connected together so as to achieve a desired function such as mixing of a sample, moving the sample through the device, reacting the sample with different reagents, and so on. These chips may be inserted into machines to control the performance of a test and measure the results.
However, it has been found that handling a sample in a microfluidics device can be very difficult. In such small channels as are found on a conventional LOC, it is difficult to apply external forces to move the sample from one site to another to perform different actions on the sample. There is also a limit to the complexity of a LOC device which operates purely using capillary action. Furthermore, owing to the small sample sizes of LOC's, the devices have reduced sensitivity and the probability of a target being present in the sample is thus reduced.
An alternative approach is to use a fluidic cartridge. The scale of the components of a fluidic cartridge is larger than for a microfluidic device, and so it becomes possible to move a sample through various different sites to perform different actions on it. This makes it possible to perform more complex tests than may be conducted using typical LOC devices, whilst still providing an analytical system of potential use in a local GP surgery.
Scientific assays useful in medical diagnostics have increasingly involved biochemical procedures, such as the polymerase chain reaction (“PCR”). The PCR assay has provided a powerful method of assaying for the presence of defined segments of nucleic acids. It is therefore desirable to perform a PCR assay on a fluidic cartridge.
Reducing PCR to the microchip level is important for portable detection technologies and high-throughput analytical systems. The method can be used to assay body fluids for the presence of nucleic acid specific for particular pathogens, such as the Chlamydia trachomatis bacterium, HIV or any other pathogenic microbe.
The introduction of commercially available automated DNA amplification assays has allowed more laboratories to introduce these technologies for routine testing of specimens. However, there is a need to improve the fluidic devices used for this purpose.
Fluidic devices are often used for sample preparation and analysis of biological or chemical liquid samples. During sample preparation, the sample typically enters through a sample input port and may pass along a main channel or into a cavity before reaching a sample chamber where it may be analysed. Additional reagents, buffers, solutions or fluids may be passed along the channel to prepare the sample for analysis. For example, when preparing a bacterial sample for PCR analysis, a lysis buffer may be used to lyse the bacteria, then a wash buffer may be passed through to wash any unwanted sample matrix through to a waste receptacle, and then the sample may be re-suspended in a final elution buffer ready for PCR amplification.
A problem arises when automating this system, since all the reagents needed in the reaction chamber for the sample analysis, such as PCR amplification, should be contained on one platform in which the sample can be inserted in a controlled operation. WO 97/16561 provides an assay system which comprises a first assembly comprising a reaction chamber, a second assembly comprising a heat source and a third assembly comprising a plurality of fluid chambers. The assemblies can move with respect to each other by sliding or translocating, so that, for example, fluid communication between the assemblies results when the first and third assemblies are adjoined. The carousel as described in Example 2 of WO 97/16561 includes seventeen fluid chambers and when rotated, these fluid chambers align with the reaction chamber so as to avoid cross contamination of the reagents. It requires that the chambers align perfectly so as to allow fluid communication and that there is sufficient volume of wash buffers and cleaning solutions to ensure trace amounts of impurities are removed.
In many biological analytical systems, in particular, in systems using amplification assays, it is important to remove certain reagents from the system prior to conducting the analysis. For example, it can be extremely important to remove all traces of the lysis buffer prior to conducting PCR amplification. Furthermore, where the microfluidic device may be re-used for a different sample it is important to ensure the device is cleaned to avoid cross-contamination. WO 2010/149995 discloses the use of cleaning solutions (such as a detergent, complexing agent, alkali or acid) or gas flushes to flush the reaction cavity prior to reuse. The air or gas may further be used to dry the channel or cavity between fluid passes. A single, overly long fluid path length requires high pressures to push the sample and reagents through the channel and also needs large volumes of cleaning solutions to ensure the whole length of the channel is cleaned effectively to avoid cross-contamination risks between fluid passes.
An alternative design, to avoid using a single fluid channel is described in WO 03/078065 and WO 2009/108260 and uses a main channel with several intersecting channels. These channels may be connected to separate input ports, offset from the main channel, and provide an alternative design for fully automated systems wherein all the reagents are contained on the microfluidic device at the start of use. The channels may alternatively connect to waste reservoirs and pass through analysis regions such as in WO 03/078065. WO 2009/108260 describes flushing cleaning solutions through the main channel to clean the microfluidic circuit and using air to push the sample through the main channel.
The introduction of secondary channels intersecting a main channel, whilst beneficial in introducing different fluids or reagents through separate, distinct channels, will always create ‘dead-legs’. A dead-leg is a section of the channel through which fluid does not flow, and is considered to be a source of contamination. They are often found where a side channel intersects a main channel since fluid can accumulate at or near the intersection point, at the dead-leg, and remain there until the next fluid pass, adding a contamination risk.
For instance, FIG. 16 shows a known microfluidic junction B110, an outlet channel B111, and a plurality of circuit units B112, B113, B114. A microfluidic junction B110 is an area for converging multiple fluids. An outlet channel B111 is capable of receiving fluid from the microfluidic junction B110. An outlet channel B111 includes a first end connected with the microfluidic junction B110, a second end connected with a waste reservoir B115, and an analysis region B116 positioned between the first end and the second end of the outlet channel B111. Each circuit unit includes a source channel B117 with a first end capable of receiving sample fluid and a second end connected with the microfluidic junction B110; a branch channel B110 connected with the source channel B117 at an intersection B119; and a flow diversion system capable of differentially directing fluid flowing through a source channel either into the microfluidic junction B110 or into a branch channel B118. The branch channel B118 is further connected to a waste reservoir B120. When fluid enters into the circuit units B112, the fluid flows along the source channel B117, to the intersection B119 and is directed to either continue through the source channel B117 and into the microfluidic junction B110, or into the branch channel B118. Fluids that are directed toward the junction B110 converge in the junction B110 and flow into the outlet channel B111, through the analysis region B116 and into the waste reservoir B115.
In FIG. 16, several dead-legs B108 exist at the intersection of the branch channels B118 with the source channels B117, and at the intersection of the source channel B117 with the outlet channel B111. FIG. 17 further illustrates a series of branch channels B101, B102, joining a main channel B100 and the presence of dead-legs B108 in the branch channels B101 and B102 at or near these intersection points.
It is very difficult to remove the presence of dead-legs completely. Special valves can be designed to reduce the dead-leg, but there is still a small but finite volume associated with these valves which may allow for fluid accumulation. Accordingly, there is a need in the art to provide a microfluidic device which allows for the addition of reagents from secondary channels to the main channel without cross-contamination.
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