Abstract:
Improved detection and enumeration of magnetic particles in a flowing stream by enveloping the particle-containing sample stream with buffer streams from the sides and from the top, thus individualizing the particles and navigating the sample stream as a single-file flow into the proximity of sensors embedded underneath the flow channel. At the same time, larger physical size of the flow channel alleviates problems such as channel clogging. Magnetic particles can represent any analyte of interest, such as biomolecules or bacterial cells, which are labeled with magnetic labels.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to detecting and enumeration of small magnetic particles flowing in a stream and to methods and instruments to enhance that detection. 
       BACKGROUND OF THE INVENTION 
       [0002]    This invention relates to an improvement over an earlier Porter et al. invention, U.S. Pat. No. 6,736,978 of May 18, 2004. The disclosure of that patent is incorporated herein by reference. 
         [0003]    In recent years there has been an increasing interest in magnetic labels for chemical and bioanalysis, as exemplified by the interest in immunomagnetic separation technology, which is a proven method for such tasks as monitoring parasites in raw surface water. In some examples, the requirements of microorganism filtration, concentration, separation, and monitoring require bulky instrumentation and manual operation. Furthermore, magnetic tags can be used as separators for detection of molecules or cells of interest. One such example is described in Kriz; C. B.; Radevik, K; Kriz, D. “Magnetic Permeability Measurements in Bioanalysis and Biosensors,” Anal. Chem. 1996, 68, 1966, in which a ferromagnetic sample is placed in a container which in turn is placed in a measuring inductor electrically connected in a bridge sensing circuit. 
         [0004]    In U.S. Pat. No. 6,736,978 there was provided a method of monitoring analyte flowing in fluid streams. A giant magnetoresistive sensor (GMR) had a plurality of sensing elements that produce electrical output signals; the signals vary dependent on changes in the magnetic field proximate the elements. A stream including the analyte was provided, and the stream had a magnetic property that was dependent on the concentration and distribution of analyte therein. The magnetic property was imparted by use of ferromagnetic particles or by use of paramagnetic or superparamagnetic particles in conjunction with application of a magnetic field. The stream flowed past the giant magnetoresistive (GMR) sensor in sufficiently close proximity to cause the magnetic properties of the stream to produce electrical output signals from the GMR. Electrical signals were then monitored as an indicator of the analyte concentration or distribution in the stream flowing past the GMR. 
         [0005]    The apparatus for practicing that method included a giant magnetoresistive (GMR) sensor having a plurality of sensing elements for detecting localized changes in the magnetic field proximate the elements. Microfluidic channels were associated with the GMR sensor closely proximate the elements of the sensor. The proximity was such that the paramagnetic particles flowing in the channels caused an output from the GMR sensor that was indicative of the concentration or distribution of magnetic particles. A source of analyte in the fluid stream was altered such that the fluid stream had a magnetic property that was related to the concentration or distribution of the analyte in the stream. The fluid source was connected to the microfluidic channels for flowing a stream including the analyte past the GMR sensor. An electrical monitor was responsive to the GMR sensor for measuring and recording changes in the output signal as an indication of the magnetic properties and therefore analyte concentration or distribution in the stream flowing past the GMR sensor. 
         [0006]    It has been determined that key to the success of the device and method described in the earlier Porter et al. patent is the efficiency of flow in the microfluidic channels and the efficient focusing of the flow stream close to the detector to improve detection. In particular in the present improvement on the prior patent the microfluidic layout of the device incorporates a novel three-dimensional fluidic focusing scheme that is easy to accomplish through two simple standard fabrication steps. This improvement in the device of the prior invention is the primary objective of the present invention. 
         [0007]    In a more general sense, another primary objective is a magnetic particle detection arrangement that provides for better detection of passing magnetic particles in a liquid flow and entraining them to enhance detection capability while reducing the risk of microfluidic channel clogging. 
         [0008]    Another objective is to develop a system useful with any microfabricated magnetic sensor, such as magnetic tunneling junctions or Hall sensors. 
         [0009]    The method and means of accomplishing the above objectives and advantages of the invention will become apparent from the following detailed description when taken in conjunction with the accompanying drawings. 
       BRIEF SUMMARY OF THE INVENTION 
       [0010]    The method and device for enhanced detection of analyte in flowing fluid streams past a GMR sensor by utilizing a unique sample flow stream, preferably narrowed to a pinch flow configuration at one point and also confined towards the channel bottom by a vertical focusing stream that enters from the top of the sample flow and squeezed laterally as well by a lateral entry focus stream. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]      FIG. 1  is a conceptual drawing of how the three dimensional focusing scheme can be implemented by the geometry of microfluidic channels as used in the present invention. 
           [0012]      FIG. 2  shows the fluidic layout to accomplish vertical flow focus and the lateral flow focus of the main channel particle stream of a microfluidic channel piece in order to enhance detection. 
           [0013]      FIGS. 3   a  and  3   b  show compiled results from two dimensional numerical modeling of the flow focusing architectures for the lateral flow pinch and for the vertical confinement flow stream, both as illustrated in  FIGS. 1 and 2 . 
           [0014]      FIG. 4  shows graphically how an initially uniform rectangular array of sample stream lines is refocused when passing through the microfluidic channels used in the present invention. 
           [0015]      FIGS. 5   a  and  5   b  shows the GMR signals when the device is used in accordance with the example described for the present invention. 
       
    
    
       [0016]    While the invention will be described in connection with certain preferred embodiments, there is no intent to limit it to those preferred embodiments. Rather the intent is to cover all alternatives, modifications and equivalents as included within the spirit and scope of the invention as defined by the appended claims. 
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0017]    The general layout of a multiple element GMR flow cytometer is shown in  FIGS. 1 ,  2  and for the state of the art in my prior patent. In the present unit and methodology therefore microfluidic channels of the prior unit are replaced with new ones. The microfluidic layout of the present device is illustrated in  FIG. 1 . The conceptual drawings of the three dimensional focusing scheme  10  is shown. The sample stream  12  is first narrowed in a “pinch flow” configuration  14  and then confined towards the channel bottom  16  by the combination of a vertical focusing stream  18  that enters from the top  20  of the sample flow channel  22 . Stream lines  24  and sheath flow boundry  25  are shown to visualize the change of the sample stream boundaries. 
         [0018]    The concept of three dimensional focusing scheme as shown in  FIG. 1  was developed as a result of recognition of two key issues identified in a recent report that critically examined the design and performance challenges in the development of a GMR flow cytometer. First, the sensor size (detection region  24  of  FIG. 2 ) needs to be in the range of several micrometers to make single cell detection possible. Moreover, the proximity of the target to sensor is crucial, since observed field decays with the third power of distance. These requirements necessitate precise navigation of the sample stream over sensors. Simply pumping the sample through channels with small cross-sections can pose two problems: 1) clogging in the presence of particles with a distribution of sizes or particle aggregates, and 2) fabrication techniques become more challenging with downscaling the feature sizes. The inventors therefore chose to develop a detection format where micrometer-sized GMR sensors are buried beneath the channel of a relatively large cross-section. Upstream from sensors, the sample stream is hydrodynamically focused by a buffer flow from the sides  15  and from top  18  and  20  to form a narrow and thin flow stream  24  along the central bottom portion  16  of the channel (see  FIG. 1 ). The sense elements in sensor bridges of the device (detector region  24 ) are centered along the projection of the sample stream after completing both focusing steps so that the main sample stream  12  is configured as in  FIG. 4 . 
         [0019]    Since sample flow is confined and focused by surrounding and collapsing the sample stream with fluid rather then solid constrictions, this approach greatly reduces the risk of channel clogging. To ensure that target particles traverse precisely the sensor field of view, the flow rates from the two branches of the lateral-focus system need to be carefully balanced. Generally, the flow rates of the side-flow and vertical-flow buffer streams are chosen and controlled to ensure that the focused sample stream width, height, and the position in the main channel are optimal for a given size and geometry of the GMR sensors. Most times they are within 20% of each other and preferably within 10%. Those skilled in the art can readily make the necessary adjustments through trial and error experience. 
         [0020]    A similar model for the case of imbalanced vertical focus flow rates (±10% deviation) shows only a slight drift of the sample stream from the middle of the channel (x=0). To achieve balanced flow rates and to reduce the number of fluidic connections to the chip, the lateral and the vertical focus feed from two external pumps are each split into two streams on-chip, immediately following the inlet ports. A careful design of the overall fluidic structure, including channel constrictions that serve as fluidic ballast resistors, ensures symmetrical backpressures and therefore symmetrical flows in the focusing branches.  FIG. 4  gives an overview of two fluidic designs, and examples of two different sensor arrangements. 
         [0021]      FIG. 2  demonstrates the fluidic layout corresponding to the schematics of  FIG. 1  as occurs in the microfluidic channels associated with the GMR for providing the microfluidic channels closely proximate the sensor element  25   a  of the device described in our previous patent. In particular, the sample flow through the detection region  25   a  is oriented along the y-x axis and perpendicular to the applied field. The sample is sent through the vertical inlet  26  (depicted as an arrow). There is a lateral focusing inlet  28  (also depicted as an arrow) for enveloping the main particle sample fluid stream  30  from the side as well as the vertical sample inlet  26  for enveloping the main particle fluid stream  26  from the top. After being shaped and directed by the stream from the lateral focusing inlet  28  and the top or vertical focusing inlet  26 , sample stream  30  is focused in region  32 , then passes through the detection region  25 . 
         [0022]    The system utilizes spin-valve GMR sensors that are fabricated as 30-μm long, 2-μm wide strips, and they are sensitive to the transverse component of the magnetic field (x-axis direction). The electrically active area that generates the electrical signal is defined by the positioning of the electrical contacts on the sensor. The sensors are arranged in a Wheatstone bridge configuration. In the case of one sense resistor (R s ) and three reference resistors (R r ), the measured signal is given by Equation 1: 
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         [0000]    where E bias  is the constant bias voltage supplied to the bridge. In case of a pair of adjacent sense resistors, as in  FIG. 2 , assuming that the fringe field of the target is uniform across the overall sense resistor area, the measured signal will be twice as large as that obtained from Equation 1. The choice of the electrical layout and sensor size will depend on the size of the target, and will also dictate the choice of the fluidic layouts [ FIG. 2 ] used to achieve the focused flow. 
         [0023]    In our development work we studied how sample stream dimensions scale with the sample-to-focus flow rate ratios. In some cases, a simple linear approximation provides acceptable results. Hofmann et al. briefly discussed the experimentally observed non-linear scaling of sample thickness in vertical confinement flows, Hofmann, O., Niedermann, P. &amp; Manz, A., “Modular approach to fabrication of three dimensional microchannel systems in PDMS-application to sheath flow microchips”,  Lab on a Chip  1, 108-114 (2001). In the following discussion, we give a more thorough analysis that reveals important phenomena that somewhat contradict day-to-day linear intuition. The non-linear behavior is a consequence of the coupling of mass conservation requirements with the parabolic flow velocity profiles present in microchannels. The finite-element models were developed in FEMLAB based on fluidic layouts described above, and estimation of the experimental parameters—flow rates, target concentration range and the velocity distribution, and requisite sampling rates. 
         [0024]      FIGS. 3   a  and  3   b  show results from two-dimensional models of the lateral and vertical focusing junctions. We define a relative flow rate as a ratio of the volumetric flow rate of the sample stream to the total volumetric flow rate. The models show that in case of the symmetrical “pinch-flow” focusing [ FIG. 3(   a )], the sample stream width follows a linear correlation with the relative sample flow rate, at least up to a relative flow ratio of 0.33. The sample-stream width, however, is narrower than that predicted by the simple rationing of flow rates, as the focus streams widen in order to compensate for their lower flow velocities in the parabolic flow profile. In case of the vertical confinement flow [ FIG. 3  ( b )], a non-linear dependence is readily apparent, and arises because the sample stream confined to the vicinity of the channel bottom compensates for the lower flow velocity by forming a thicker layer. This situation means that the actual sample-stream thickness will always be higher than that predicted by simple rationing. 
         [0025]    These results can be used to estimate the relative flow rates required to achieve a given size of the sample stream. For example, in case of 2×10 μm sensors, with flow parallel to the field (x-axis direction), the sample stream width should be around 10 μm, i.e., a Δy of 0.2, relative to the channel width of 50 μm. This width value translates into the relative sample flow rate of around 0.3, or ωs/(ωs+ωl)=0.3, calling for the lateral focus flow rate of ωl=2.3 ωs. In a similar way, to achieve a thickness of at least 3 μm, or Δz=0.1 relative to the channel depth, the required vertical-focus flow rate is ωv=107 ωs, yielding a total flow rate of ω total=110 ωs. To understand the significance of the parabolic flow profiles in non-asymmetric flow focusing systems, it is useful to compare the above factor of 110 to the ratio of the channel cross-section to the focused stream cross-section, which equals 50 in this example. 
         [0026]    Further analysis of three-dimensional models reveals another non-linear effect (FIG.  4 )—a distortion of the sample streamlines upon focusing. The outcome is interesting and worth noting, since it suggests that an initially homogeneous suspension of target particles will tend to exhibit a slightly higher concentration in the top region of the focused sample stream. 
         [0027]    That is to say an initially uniform rectangular array of the sample streamlines maps non-uniformly into a focused sample stream as illustrated in  FIG. 4 . The initial array upstream from the focusing junctions consisted of 5×6 streamlines and covered the sample inlet cross-section. Upon focusing, the streamline array is distorted as illustrated in  FIG. 4 , with the streamline density higher in the top  34  region than the bottom  36 . 
         [0028]    The following example is shown to demonstrate the efficiency of the unit in the detection of magnetotactic bacteria. 
       EXAMPLE 
     Detection of Magnetotactic Bacteria 
       [0029]    Cells of marine magnetotactic vibrio MV-1 were cultured and then fixed overnight at 4° C. in 0.1% glutaraldehyde. The cells were washed three times, resuspended in Tris-borate buffer (pH 8.0), and subsequently stained by adding the Syto 16 fluorescent dye (1 mM solution in dimethylsulfoxide) to a final dye concentration of 2 μm. Fluorescent staining enabled determination of the cell counts using a hemocytometer and a microscope with a 40× objective lens, and the suspension was diluted with Tris-borate buffer to a final concentration of 30 000 cells/μL. The labeled cells appeared well dispersed, with no noticeable aggregates. Three syringe pumps were used to deliver the sample suspension and the two focusing streams (Tris buffer) to the chip. The device was based on the y-direction flow layout (perpendicular to the field), and featured bridges with a single sense and three reference 2×2 μm sensors. Only one of the four bridges was functioning properly. Applied bias voltage was E bias =+0.2 V which required a current of 3.3 mA. The signal was digitized at 100 kHz after 50-fold amplification. 
         [0030]    Because of the misalignment of the fluidic layer relative to sensors, a narrowly focused sample stream would miss the detection volume above the sensor. The flow rates were therefore chosen to produce a relatively wider sample stream that partly flows over the sense element. The flow rates were: sample flow rate ωs=0.023 μL/min, total lateral focus flow rate ωl=0.07 μL/min, and total vertical focus flow rate ωv=0.5 μL/min. These parameters yield a 9×9 μm cross-section of the sample stream. The collected GMR signal is shown in  FIG. 5 . When the lateral focus flow rate was increased to 0.13 μL/min to narrow down the sample stream to about 5 μm, the GMR signal collapsed to the baseline noise level. This was expected, since under those conditions the sample stream would flow just at the side of the detection volume. 
         [0031]      FIGS. 5   a  and  5   b  show a snapshot of the GMR signal recorded during the flow of the magnetotactic bacteria, with  5   b  showing a detail of the same data set. External field strength equaled 1800 Am −1  (22.6 Oe). 
         [0032]    Preliminary experimental findings, based on magnetotactic bacteria as targets, apparently demonstrate single-cell detection events. Further work is needed to substantiate this and to fully exploit the potential of the system; this test however is sufficient to demonstrate that the focused flow stream cause by impinging the main particle stream from the top and the sides just prior to entering the detector focuses the flow stream close to the detector and improves detection significantly. Potential for clogging risk is further reduced and one can count the particles one at a time without them sticking to the walls. 
         [0033]    It can therefore be seen that the use of the microfluidic chip or channel to direct the stream as illustrated herein accomplishes at least all of its stated objectives.