Patent Description:
Patent document published <CIT> discloses a microfluidic injector with a cassette device that is used for the isolation, amplification, and identification of DNA or protein. The cassette-based device includes a series of channels and chambers that are used add sample, reagents, diluents, and perform reactions (amplification and digestion). The cassette-based device also discloses the use of capillary electrophoresis tubes/gel (<NUM>) that is used to carry out gel electrophoresis on the DNA/protein digestion products.

Capillary electrophoresis (CE) is a well-known chemical separation technique, which has the advantages of high efficiency, high resolution, low consumption of sample and reagent, and simple instrumentation compared to other conventional separation methods, such as high-performance liquid chromatography (HPLC). (See, e.g., <NPL>; and <NPL>).

Unlike HPLC, CE can also be readily miniaturized into microfluidic chips. (See e.g., <NPL>; and <NPL>.

"Microchip electrophoresis" (MCE) is especially important for applications that require portability, compactness, or low instrument cost. CE and MCE are employed in diverse applications including DNA and protein separation, detection of disease biomarkers, environmental pollutant monitoring, food (e.g. wine) analysis, and pharmaceutical analysis. (See, e.g., <NPL>; <NPL>; <NPL>; S. Ruokonen, F. Lokajová, I. Kilpeläinen, A. King, and S. Wiedmer, "Effect of ionic liquids on the interaction between liposomes and common wastewater pollutants investigated by capillary electrophoresis," J. Chromatogr. <NUM>-<NUM>, Jul. <NUM>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>.

CE and MCE, however, have often been considered to have inferior reproducibility compared to other separation techniques such as HPLC or gas chromatography (GC). Thus, there has not been as widespread use of CE or MCE in quantitative analysis. (See, e.g., <NPL>; and <NPL>).

Numerous advances have largely eliminated this concern in recent years, though achieving the desired degree of reproducibility (e.g. peak area relative standard deviation (RSD) < <NUM>%) remains a challenge in many cases. (See, e.g., <NPL>; <NPL>; <NPL>).

In MCE, both electrokinetic and hydrodynamic injection modalities have been extensively studied, each with their own advantages and disadvantages. In electrokinetic injection, the amount injected depends on the applied potential and injection time. This method can suffer from injection bias, however, due to the different electrophoretic mobility of different species, making accurate quantitation of impurities very difficult. To inject a sample plug that is representative of the original sample, hydrodynamic (pressure-driven) injection has been used, in which a valve or pump is actuated for a certain time to control the amount injected. Relative standard deviation (RSD) of peak area in the range of <NUM> to <NUM>% has been reported. (See, e.g., <NPL>; <NPL>). However, the consistency of injection from sample to sample can still be affected in this approach by sample viscosity , stability of pressure source, and potential variation in the response time of micro-valves, etc. (See, e.g., <NPL>).

Generally, the invention described herein focuses on improving the sample injection process to achieve high sample injection repeatability to the level needed for quality control (QC) testing of short-lived radioactive positron emission tomography (PET) tracers and other applications. To further improve the consistency of injection suitable for chemical purity analysis, a novel volumetric micro-injector (also referred to as a microfluidic injector) for capillary electrophoresis (CE) for highly repeatable sample injection has been developed which eliminates known biases in electrokinetic and hydrodynamic injection. The micro-injector chip may be made of poly(dimethylsiloxane) (PDMS). The micro-injector chip contains a channel segment (i.e., a microfluidic injecting channel) with a well-defined volume. Similar to the operation of an HPLC injection valve, the microfluidic injecting channel serves as an injection loop. Using a series of on-chip located micro-valves, the microfluidic injecting channel can be connected to a sample source during a "loading" step, and to a CE separation channel during the "injection" step. For the injection step, the valves along the CE flow path are opened and electrophoretic potential is applied to separate the sample.

Accordingly, one embodiment of the present invention is directed to a micro-injector (microfluidic chip) for capillary electrophoresis, according to claim <NUM>. As used herein, the term "micro-injector" and "microfluidic" refer to devices configured to handle small amounts of sample fluids having volumes of less than 100nL (nanoliters), and which have fluid handling channels having a width of less than <NUM>, or less than <NUM>. Further developments of the invention are according to claims <NUM>-<NUM>.

Another embodiment of the present invention is directed to a method of using the microfluidic injector, according to claim <NUM>. Further developments of the invention are according to claim <NUM>.

The method of using the microfluidic injector may also include any combination of the additional aspects of the microfluidic injector, as described above.

Another embodiment of the present invention is directed to a method of using the microfluidic injector, according to claim <NUM>. Further developments of the invention are according to claims <NUM> and <NUM>.

A further embodiment of the present invention is directed to a method of using the microfluidic injector, according to claim <NUM>. Further developments of the invention are according to claim <NUM>.

The foregoing and other aspects of embodiments are described in further detail with reference to the accompanying drawings, wherein like reference numerals refer to like elements (e.g., elements having the same number are considered like elements, such as 50a and 50b) and the description for like elements shall be applicable for all described embodiments wherever relevant.

<FIG> illustrates a CE system <NUM> which utilizes a microfluidic injector <NUM> for capillary electrophoresis (CE), according to one embodiment of the present invention. The microfluidic injector <NUM> is configured to handle a buffer fluid and sample fluid to isolate a plug of sample having a defined volume and inject the plug of sample into a separation device <NUM>. As described in more detail below, the microfluidic injector includes a plurality of microfluidic channels <NUM>, micro-valves <NUM> (referred to as "valves <NUM>") disposed in the channels <NUM>, and control channels <NUM> (see <FIG>) each operably coupled to a respective valve <NUM> for controlling the operation of the micro-valves <NUM>. The separation device <NUM> may be a fused silica capillary <NUM> (<NUM> in length and <NUM> I. ) as shown in the illustrated embodiment, or other suitable electrophoresis separation device.

The CE system <NUM> includes a series of computer-controlled valves <NUM> connected to each of the control channels <NUM> in order to control the actuation of the micro-valves <NUM>. In the described embodiments, the micro-valves <NUM> are fluid pressure actuated valves and the computer-controlled valves <NUM> selectively control fluid pressure to the micro-valves <NUM> in order to open and close the micro-valves <NUM>. Hence, in the described embodiment, the computer-controlled valves <NUM> may be solenoid valves, or other suitable valve for selectively controlling a fluid pressure. Each of the computer-controlled valves <NUM> has a respective inlet connected to a gas pressure source <NUM> and a respective outlet connected to a respective one or more of the control channels <NUM>. The gas pressure source <NUM> may be shop compressed air or a pressurized nitrogen supply, or other suitable source of gas pressure. The control channels <NUM> may be filled with water or other liquid in order to remove air from the control channels <NUM> which can permeate through the substrate <NUM>. The computer-controlled valves <NUM> are operably coupled to a digital interface module <NUM>. The digital interface module <NUM> is operably coupled to a computer <NUM>, such that the computer <NUM> and digital interface module <NUM> can control the actuation of the computer-controlled valves <NUM>, and in turn, the actuation of the micro-valves <NUM>.

Still referring to <FIG>, the CE system <NUM> has a sample source <NUM> (e.g., a sample vial or other container filled with sample) containing a fluid sample connected to a sample inlet channel 14a of the micro-injector <NUM>, and a buffer source <NUM> (e.g., a buffer vial or other container filled with buffer) containing a fluid buffer solution connected to a buffer inlet channel 14b of the micro-injector <NUM>. The sample source <NUM> is also connected to a first pressure regulator <NUM> which is connected to the gas pressure source <NUM>, so that sample can be injected into the sample inlet channel <NUM>. Similarly, the buffer source <NUM> is connected to a second pressure regulator <NUM> which is connected to the gas pressure source <NUM> so that buffer can be injected into the buffer inlet channel <NUM>.

The separation device <NUM> has an inlet end <NUM> connected to a capillary port <NUM> (outlet port) of the microfluidic injector <NUM>, an outlet end <NUM> connected to a waste well <NUM>. The waste well <NUM> may be a PDMS-based waste well, and may be located on the same substrate <NUM> forming the microfluidic injector <NUM> or a different substrate ("chip").

In some embodiments, the separation device <NUM> is provided using a microfluidic capillary channel formed in, or disposed on, the same substrate <NUM> of the microfluidic injector <NUM>, for example, within a separation channel, or other channel formed on the substrate <NUM>.

The CE system <NUM> also includes a power supply <NUM> coupled to a computer-controlled voltage relay <NUM> for selectively applying a voltage across an injection channel 14c of the microfluidic injector <NUM> and the separation device <NUM>. The voltage applied by the power supply <NUM> provides the electrophoretic potential for driving a fixed volume of sample from the injection channel 14c into and through the separation device for separation and detection. The voltage relay <NUM> has a first electrode <NUM> which is coupled to an injector buffer well <NUM> of the microfluidic injector <NUM>, and a second electrode <NUM> which is coupled to a ground <NUM> and the waste well <NUM> to complete the electrical circuit. A current meter <NUM> is connected to the electrical circuit to measure current in the circuit and interfaces with the computer <NUM> and/or digital interface module <NUM>. Separation detection within the separation device <NUM> is accomplished using a light source <NUM> (e.g., a deuterium light source) and a spectrophotometer <NUM>. Mounting devices, such as sleeves, are mounted on the separation device <NUM> for mounting a light source fiber coupled to the light source <NUM> and a detection fiber coupled to the spectrophotometer <NUM>. The spectrophotometer <NUM> measures absorbance of light from the light source <NUM> as a sample passes through the separation device <NUM>. The spectrophotometer <NUM> also interfaces with the digital interface module <NUM> such that measurements obtained by the spectrophotometer <NUM> are transmitted to the computer <NUM> via the digital interface module <NUM>.

Turning to <FIG>, three different embodiments of the microfluid injector <NUM> of the CE system <NUM> are illustrated. The three different microfluidic injectors <NUM> have many of the same or substantially similar features such that same or similar elements in each of the three embodiments have like reference numerals and the description with respect to one of the embodiments applies to the other embodiments. Referring first to the embodiment illustrated in <FIG>, the microfluid injector 12a comprises a substrate <NUM> such as a chip or multi-layer chip, in which the fluid sample handling elements are formed. The fluid handling elements include the injection channel 14c, the buffer well <NUM>, the capillary port <NUM>, the sample inlet channel 14a (first inlet channel 14a), the buffer inlet channel 14b (second inlet channel 14b), and a first waste channel 14d. The microfluidic injector <NUM> also includes an optional auxiliary waste channel 14f. The control channels 18a-18f are also formed in the substrate <NUM>.

The injection channel 14c has a first end connected to the buffer well <NUM> and a second end connected to the capillary port <NUM>. The sample inlet channel 14a, buffer inlet channel 14b, first waste channel 14d and optional, auxiliary waste channel 14f are each connected to the injection channel 14c at different locations of the injection channel 14c.

Selectively controllable micro-valves 16a-16f (also referred to as "valves") are disposed in each of the channels 14a-14f. The valves <NUM> are located in the connecting channels 14a, 14b, 14d 14f (channels connecting to the injection channel 14c) just before (at or proximate to) the junction to the injection channel 14c. A first valve 16a (sample inlet valve 16a) is disposed in the sample inlet channel 14a, a second valve 16b (buffer inlet valve 16b) is disposed in the buffer inlet 14b, a third valve 16d (first waste channel valve 16d) is disposed in the first waste channel 14d, a fourth valve 16c (first injection channel valve 16c) is disposed in the injection channel 14c upstream of the junction of the injection channel 14c to the connecting channels 14a, 14b, 14d, 14f (proximate an upstream end of the injection channel, a fifth valve 16e (second injection channel valve 16e) is disposed in the injection channel 14c downstream of the junction of the injection channel 14c to connecting channels 14a, 14b, 14d, 14f (proximate a downstream end of the injection channel 14c), and a sixth valve 16f (auxiliary waste channel valve 16f) is disposed in the auxiliary waste channel 14f. The terms "downstream" and "upstream" as used herein are relative to the flow of fluid through the injection channel 14c from an upstream end of the injection channel 14c to a downstream end of the injection channel 14c connected to the output port <NUM>. The fourth valve 16c is positioned along the injection channel 14c such that closing the fourth valve 16c seals the buffer well <NUM> from the sample inlet channel 14a, buffer inlet channel 14b and first waste channel 14d. The fifth valve 16e is positioned along the injection channel 14c such that closing the fifth valve 16e seals the capillary port <NUM> from the sample inlet channel 14a, buffer inlet channel 14b and first waste channel 14d. The portion of the injection channel 14c between the fourth valve 16c and the fifth valve 16e forms a first sample chamber <NUM> having a first defined volume. A volume of sample having the first defined volume contained in the first sample chamber <NUM> by isolating the volume of sample between the fourth valve 16c and the fifth valve 16e is referred to as a "sample plug" or "plug of sample.

The micro-valves <NUM> may be any suitable controllably actuatable valve. For example, the micro-valves <NUM> may be pressure-actuated valves, such as push-up valves <NUM> which utilize so-called push-up valve architecture. In push-up valves, the microfluidic channel <NUM> that carries the flow of fluid has a flexible diaphragm or membrane <NUM> that is actuated by the respective, separate control channel <NUM> (shown in <FIG> connected to each valve <NUM>). <FIG> illustrates a schematic view of one embodiment of a push-up valve <NUM>. For example, a fluid that is driven or pressurized by compressed gas such as air that is controlled via the computer-controlled valves <NUM> can be used to selectively actuate the valve <NUM> causing deflection of a diaphragm or membrane <NUM> to "close" the microfluidic channel <NUM> to fluid flow. The air or gas may be used to drive another fluid such as water that expands the flexible diaphragm or membrane <NUM> when the actuation or control channel is actuated. The valve <NUM> can be relaxed and returned to its "open" state by not applying, or releasing, the pressure of the actuation fluid in the control channel <NUM>.

As shown in <FIG>, a respective one of the control channels 18a-18f is operably coupled to a respective one of the valves 16a-16f. As explained above, a respective computer-controlled valve <NUM> is connected to each of the control channels 18a-18f in order to independently control the actuation (i.e., the opening and closing) of a respective one of the micro-valves <NUM>.

In the illustrated embodiment of <FIG>, the substrate <NUM> has <NUM> layers of PDMS, a control channel layer 70a and a fluid channel layer 70b, which are bonded together and then bonded to a base layer 70c. The microfluidic channels <NUM> are formed in the fluid channel layer 70b, and the control channels <NUM> are formed in the control channel layer 70b. The substrate <NUM> may be manufactured of PDMS using a multilayer soft lithography process. In this molding process, a mold for each layer 70a, 70b is fabricated using a photolithographic process. The molds are then used to fabricate each layer 70a, 70b from PDMS. In one exemplary product used for the experiments described below, a mold for the fluid channel layer 70b was fabricated from a <NUM>" silicon wafer using a SU-<NUM><NUM> negative photoresist, and a mold for the control channel layer 70b was fabricated from a second <NUM>" silicon wafer using a SPR <NUM>-<NUM> positive photoresist. The control <NUM> channels were <NUM> deep and <NUM> wide and were rectangular in cross section. The microfluidic channels <NUM> were <NUM> deep and <NUM> wide and were rounded in cross section to enable complete channel sealing when the respective valves <NUM> were actuated to a closed state. The first defined volume (i.e., sample plug volume) within the first sample chamber <NUM> was designed to be about <NUM> nL in volume (other volumes could also be formed, such as from 2nL to 4nL, from 2nL to 6nL, from 3nL to 6nL, from 1nL to <NUM> nL, from <NUM>. 1nL to 100nL less than 6nL, or less than 10nL). Once the control channel layer 70a and fluid channel layer bonding was complete, the chips were removed from the control wafer mold. A manual press equipped with metal punches was then used to create access to channel for fluidic and control lines, and an output port for a fused silica capillary. Teflon coated fused-silica capillary with a total length of <NUM> was inserted into the PDMS injector chip. The silica capillary may be "sharpened", e.g. via mechanical sanding/polishing prior to insertion into the channel. The combined chip (fluid and control layers) was then bonded to the flat PDMS substrate using a corona discharge. The other end of capillary is positioned to be submerged in buffer solution inside the separate waste reservoir also made with PDMS.

As shown in <FIG>, the microinjector 12a is connected to the separation device <NUM> with a perpendicular junction geometry in which the longitudinal length of the microinjector <NUM> and the injection channel 14c are oriented perpendicular to the separation device <NUM>. The perpendicular geometry results in significant dead volume at the junction of the microinjector <NUM> and the separation device <NUM>. For a microinjector 12a having the scale as described above, the dead volume is estimated to be about <NUM>-<NUM> nL. This dead volume is likely to result in significant dispersion within a sample plug that could lead to peak broadening and tailing contrast.

In order to minimize the dead volume, and resulting dispersion within a sample plug, resulting from the perpendicular junction geometry, another embodiment of a microfluidic injector 12b according to the present invention is illustrated in <FIG>. The microfluidic injector 12b is the same as the microfluidic injector 12a shown in <FIG>, and described above, except that the microfluidic injector 12b is connected to the separation device <NUM> with a collinear junction geometry in which the longitudinal length of the microinjector <NUM> and the injection channel 14c are oriented collinear with the separation device <NUM>. The collinear geometry of the microfluidic injector 12b results in a much smaller dead volume at the junction of the microfluid injector <NUM> and the separation device <NUM> than the perpendicular geometry. Indeed, the microfluid injector of Fig. 12b has nearly zero dead volume. The fabrication, operation and use of the microfluidic injector 12b is the same as the microfluidic injector 12c.

Turning to <FIG>, another embodiment of a microfluidic injector 12c according to the present invention is shown. The microfluidic injector 12c is substantially the same as the microfluidic injectors 12a, 12b, except that it is further configured to be capable of isolating a plug of sample of differing defined volumes. The substrate <NUM> of the microfluidic injector 12c further comprises a second waste channel <NUM> connected to the injection channel 14c between the fourth valve 16c and the fifth valve 16e. A seventh valve <NUM> (second wasted channel valve <NUM>) is disposed in the second waste channel <NUM>, and an eighth valve <NUM> (third injection channel valve <NUM>) is disposed in the injection channel 14c just before (at or proximate to) the junction to the injection channel 14c. A second sample chamber <NUM> having a second defined volume is formed in the injection channel 14c between the eighth valve <NUM> and the fourth valve 16c. As the second sample chamber <NUM> is only a portion of the first sample chamber <NUM>, the second defined volume is less than the first defined volume. This feature allows a sample to be loaded into the injection channel 14c between eighth valve <NUM> and the fourth valve 16c wherein the plug of sample has the second defined volume.

In still another embodiment in which a plug of sample of differing volumes may be isolated may utilize a plug generator same or similar to the device and method described in <NPL>). The plug generator contains two adjacent chambers, each of which has a volume that can be digitally adjusted by closing selected microvalves. By configuring the injection channel with additional microvalves to resemble one such adjustable-volume chamber, the micro-injector could be used to inject different sample volumes by adjusting the volume of the chamber prior to filling with sample.

Turning to <FIG>, a method of operation of the microfluidic injectors 12a, 12b, 12c to inject a plug of sample having the first defined volume into the separation device <NUM> will be described. As shown in <FIG>, various channels <NUM> of microfluidic injector <NUM> are first primed with a buffer. The second valve 16b,fourth valve 16c and fifth valve 16e are opened and the first valve 16a, third valve 16d, and sixth valve 16f are closed. The open or closed state of the valves <NUM> throughout the operation of microfluidic injector are controlled and actuated via the respective control channels <NUM> and the respective computer-controlled valves <NUM>. The auxiliary waste channel 14f is not typically utilized in the operation of the microfluidic injector <NUM>, and the sixth valve 16f remains closed through the operation of the microfluidic injector <NUM>. Optionally, the third valve 16d may be opened during the priming step in order to prime the first waste channel 14d with buffer. Buffer is injected from the buffer source <NUM> into the buffer inlet channel 14b such that buffer flows into and fills the injection channel 14c, the buffer well <NUM>, the separation device <NUM>, and optionally the first waste channel 14d.

After the microfluidic injector <NUM> is primed with buffer, the sample inlet channel 14a may be primed with sample. This may be accomplished in two ways. In a first way, as shown in <FIG>, the sample source <NUM> may be pressurized to a higher pressure (e.g., about <NUM> psi) causing any air in the sample inlet channel 14a to permeate out through the substrate <NUM> until the sample inlet channel <NUM> is filled with sample up to the first valve 16a. Alternatively, the same procedure as shown in <FIG> for loading the first sample chamber <NUM> with sample may be performed until any air in the sample inlet channel 14a has passed through the first valve <NUM> and the third valve 16d.

Next, as shown in <FIG>, the first sample chamber <NUM> is loaded with a sample. The first valve 16a and third valve 16d are opened, and the second valve 16b, fourth valve 16c and fifth valve 16e are closed. Sample is injected from the sample source <NUM> into the sample inlet channel 14a, and into the injection channel 14c and the first waste channel 14d, until sample fills the first sample chamber <NUM>. A full first sample chamber <NUM> may be confirmed by detecting that sample is exiting the injection channel 14c through the first waste channel 14d.

As shown in <FIG>, once the first sample chamber 14c is full, the first valve 16a and the third valve 16d are closed, such that a plug of sample having the first defined volume is contained in the first sample chamber <NUM> between the fourth valve 16c and the fifth valve 16e. The first valve 16a may be closed before the third valve 16d by a short time period (e.g., about <NUM>-<NUM> seconds) so that the plug of sample in the first sample chamber <NUM> is not pressurized by the sample pressure (typically about <NUM>. In other words, the sample pressure is vented through the first waste channel 14d.

Next, as shown in <FIG>, the plug of sample is injected into the separation device <NUM> by applying a voltage across the injection channel 14c and the detection device <NUM>. Before applying the voltage, the fourth valve 16c and fifth valve 16e are opened, and the first valve 16a, second valve 16b and third valve 16d are closed. The voltage causes the plug of sample to move downstream in the injection channel 14c toward the separation device <NUM>, as shown in <FIG>. As shown in <FIG>, the plug of sample is injected into the separation device <NUM> and eventually past the light source <NUM> and spectrophotometer <NUM>, as shown in <FIG>.

The operation of the microfluidic injector 12c to isolate a plug of sample in the second sample chamber having the second defined volume is similar to the operation shown in <FIG>, except for the following. During the buffer priming step of <FIG>, the eighth valve <NUM> is open and seventh valve <NUM> may be closed. Alternatively, during the buffer priming step, the seventh valve may be open in order to prime the second waste channel <NUM> with buffer. During the sample loading step shown in <FIG>, the seventh valve <NUM> is opened and the eighth valve <NUM> is closed, and sample is injected from the sample source <NUM> into the sample inlet channel 14a, and into the injection channel 14c up to the eighth valve <NUM>, and into the second waste channel <NUM>, until sample fills the second sample chamber <NUM>. A full second sample chamber <NUM> may be confirmed by detecting that sample is exiting the injection channel 14c through the second waste channel <NUM>. At the sample plug isolation step of <FIG>, seventh valve <NUM> and the eighth valve <NUM> are also closed such that a plug of sample having the second defined volume is contained in the second sample chamber <NUM> between the fourth valve 16c and the eighth valve <NUM>. Finally, in the sample plug injection steps shown in <FIG>, the eighth valve <NUM> is opened and the seventh valve <NUM> remains closed as the plug of sample is advanced through the injection channel 14c and is injected into the separation device <NUM> by applying the voltage across the injection channel 14c and the detection device <NUM>.

Using a CE system in conformance with the CE system <NUM> illustrated in <FIG> and <FIG>, a number of experiments were performed to examine the performance of the microfluidic injector <NUM> of the present invention. In a first series of experiments, multiple injections of single-species samples (i.e., <NUM> thymidine in DI water, <NUM> <NUM>'-deoxy-<NUM>'-flourothymidine (FLT) in acetonitrile-water mixture containing <NUM>% acetonitrile (v/v)) were performed for each injection method (volumetric injection and timed injection) using the same chip. These are known byproducts and products during synthesis of the PET tracer [<NUM>F]FLT.

A field of <NUM> V/cm (<NUM> VDC) was applied across the buffer well <NUM> of the micro-injector <NUM> and the waste well <NUM>/outlet end of the silica capillary <NUM> (<NUM> cm in length and <NUM> I. To detect samples in the capillary <NUM>, a detection cell having fiber optics for the spectrophotometer <NUM> and light source <NUM> was positioned <NUM> along the capillary <NUM>. Using a custom-written LabView program, absorbance was recorded as a function of time since injection of a sample plug from the microfluidic injector <NUM> into the capillary <NUM> to create an electropherogram.

Prior to use, the microfluidic channels <NUM> of the chip and capillary surface were conditioned with <NUM> NaOH for 24hr, then filled with <NUM> phosphate buffer containing <NUM> SDS. Separation proceeds by opening the ends of the flow path in the chip as described above and applying the separation voltage (4kV). As a preliminary evaluation, repeatability was measured via injection of <NUM> thymidine. Repeatability was compared for volumetric injection as well as timed-injection. For the timed-injection, the sample inlet channel 14a was used as the sample inlet and the fifth valve 16e was opened for <NUM> to introduce sample plug into separation channel. The detected UV absorbance peaks (<NUM>) in the resulting electropherograms were fit to Gaussian profile to determine retention time and area.

The performance of this new volumetric injection technique using the microfluidic injector <NUM> was compared to the common hydrodynamic injection technique of opening a micro-valve for a fixed amount of time. Measurements were repeated on three (<NUM>) different microfluidic injector chips <NUM> for each compound for each injection mode. The relative standard deviation (RSD) value of peak area was calculated for multiple runs to quantify the consistency of injected sample volume. For the viscosity study, samples consisted of <NUM> thymidine dissolved in DI water or in <NUM>% glycerol/water (v/v) were used.

The volumetric injection mode showed lower peak area RSD indicating superior injection repeatability. Volumetric injection showed in some cases better performance with a relative standard deviation (RSD) of peak area as low as <NUM>% (n=<NUM>) than the best RSD values reported in the literature for hydrodynamic micro-valve-based injection. It also showed considerably better performance than typically reported values for hydrodynamic micro-valve-based injection. Furthermore, in contrast to hydrodynamic injection, volumetric injection was found not to depend on sample viscosity.

<FIG> illustrates the electropherogram of a single run of the micro-injector <NUM> using volumetric injection. <FIG> illustrates the UV absorbance peaks of <NUM> successive runs of CE in using the microfluidic injector <NUM>, resulting in very low peak area RSD of <NUM>% indicating highly repeatable sample injection, as compared to literature reports. The same chip operated in time-dependent injection mode resulted in a higher peak area RSD of <NUM>%.

To avoid effects of buffer depletion (change in composition due to electroosmotic flow (EOF)), fresh buffer solutions were loaded manually between runs by pipette for initial experiments. This issue could instead be addressed by using larger volume buffer wells, or by using some on-chip microfluidic valves and pumps to exchange the well contents in between runs. Timed, hydrodynamic injection has an advantage that the injection volume can be changed simply by changing the time of valve opening. This can help accommodate different sized capillaries or sample concentrations. On the other hand, the volumetric chip has a fixed volume chamber and the tested design does not have the same flexibility. To increase injection volume flexibility, valve-based approaches where the length of the chamber is dynamically adjusted can be readily incorporated as has been previously shown in the embodiment of <FIG>. (See, e.g., <NPL>.

Another series of experiments was conducted to examine the ability to separate multiple compounds, and to compare results of volumetric injection (with the two different junction geometries, namely the perpendicular junction of the embodiment of <FIG> and the collinear junction of the embodiment of <FIG>) as well as the widely-used approach of electrokinetic injection. To avoid introducing additional variables, similar injection volume was used in all three different injection modes. Baseline separation of a mixture of <NUM> compounds was achieved using the microfluidic injector <NUM> with the collinear junction. <FIG> is an electropherogram of a single run of the microfluidic injector 12a having the perpendicular junction in separating a mixture of <NUM> compounds using volumetric injection. For comparison, <FIG> is an electropherogram of a single run of the microfluidic injector 12b having the collinear junction in separating a mixture of <NUM> compounds using electrokinetic injection.

Peak area RSD values for the <NUM> compounds and various injection methods are summarized in Table <NUM> shown in <FIG>. It can be seen that the capillary-to-chip junction geometry does not significantly affect the sample injection repeatability. This is expected because the injected sample amount is physically metered within the injection chamber before even seeing the junction. For volumetric injection, the peak area RSD was always < <NUM>% and values as low as <NUM>% were observed. When electrokinetic injection was performed in the same chip with the same injection volume (as verified by comparing peak areas), the peak area RSD was found to be substantially higher, indicating less consistent sample injection. From these results and the comparison of volumetric and timed injection presented earlier, it appears that sample injection repeatability is mainly influenced by the injection method.

The microfluidic injector <NUM> of the Examples described herein used a hybrid of a capillary <NUM> and a microfluidic injector chip <NUM>. In an alternative embodiment, the capillary <NUM> could be replaced by a microchannel within the microfluidic injector chip <NUM> itself. A UV absorbance detector (or other detection methods) can also be implemented directly in the microfluidic injector chip <NUM> by a variety of fabrication methods. That is the UV detector functionality may be implemented directly in the microfluidic injector <NUM> or in another chip that is coupled thereto. It is contemplated by the present invention that the microfluidic injector <NUM> could be integrated into fully-inclusive lab-on-a-chip platforms for a variety of MCE-based applications. In addition to optical detection, other modalities such as radiation detection, pH measurement and impedance measurement can be incorporated to further increase the flexibility of samples that can be analyzed.

The microfluidic injector <NUM> could be integrated into more complex lab-on-a-chip systems. For example, for QC testing of pharmaceuticals or radiolabeled imaging agents, there are a variety of other tests that are needed including sterility, pH, color, etc. It is likely that a compact device could be built, that along with MCE methods for chemical/radiochemical purity, would be able to test such samples.

The microfluidic injector <NUM> described herein offers several advantages over current methods. First, the microfluidic injector <NUM> provides for highly repeatable sample injection eliminating known sample injection biases. This volumetric microfluidic injector <NUM> for CE can eliminate variables that are still present when using hydrodynamic injection. Furthermore, bias-free precise quantitative analysis is possible independent of varying fluid properties (e.g., sample with varying viscosity). In addition, multiple successive injections of the same sample can be performed without changing the sample composition as occurs with hydrodynamic injection. The microfluidic injector <NUM> may be made using standard PDMS-based fabrication techniques. While larger scale CE platforms can attain high injection repeatability than typical MCE platforms via hydrodynamic injection. However, miniaturization is important for many applications where compactness, portability, and/or low cost are needed. Miniaturization, in fact, has even further advantages, including lower sample consumption, improved resolution, shorter separation times, improved reproducibility (e.g., from improved temperature control), and increased sensitivity and diversity of detection methods. Thus, it is desirable to boost the repeatability of MCE methods to the repeatability of traditional CE methods or better. The microfluidic injector <NUM> accomplishes this. The microfluidic injector <NUM> may be used in any CE application where quantitation and thus volume repeatability is needed, including the chemical purity analysis of pharmaceuticals and radiopharmaceuticals. Many other applications in the field of analytical chemistry where precise and reliable measurements of compound concentrations are needed could also benefit from this invention.

Claim 1:
A microfluidic injector (<NUM>) for capillary electrophoresis, comprising:
an electrophoresis separation device (<NUM>);
a substrate (<NUM>) having an injection channel (14c) having a first end connected to a buffer supply (<NUM>) and a second end connected to the electrophoresis separation device (<NUM>), a sample inlet channel (14a) connected to the injection channel (14c), a buffer inlet channel (14b) connected to the injection channel (14c), and a first waste channel (14d) connected to the injection channel (14c);
a selectively controllable first valve (16a) disposed in the sample inlet channel (14a);
a selectively controllable second valve (16b) disposed in the buffer inlet channel (14b);
a selectively controllable third valve (16d) disposed in the first waste channel (14d);
a selectively controllable fourth valve (16c) disposed in the injection channel (14c) and positioned such that closing the fourth valve (16c) seals the buffer supply (<NUM>) from the sample inlet channel (14a), the buffer inlet channel (14b) and the first waste channel (14d);
a selectively controllable fifth valve (16e) disposed in the injection channel (14c) and positioned such that closing the fifth valve (16e) seals the electrophoresis separation device (<NUM>) from the sample inlet channel (14a), the buffer inlet channel (14b) and the first waste channel (14d), and wherein a first sample chamber (<NUM>) having a first defined volume is formed in the injection channel (14c) by and between the fourth valve (16c) and the fifth valve (16e);
a power supply (<NUM>) configured to apply a capillary electrophoresis separation voltage between the buffer supply (<NUM>) and an output of the electrophoresis separation device (<NUM>) and across the injection channel (14c) so as to drive the sample located in the injection channel (14c) between the fourth valve (16c) and the fifth valve (16e) through the electrophoresis separation device (<NUM>); and
wherein the electrophoresis separation device (<NUM>) is selected from the group consisting of: a silica capillary; a capillary channel formed in the substrate; and a capillary channel disposed on the substrate (<NUM>).