阅读说明：本技术 电极断裂检测
(Electrode breakage detection
) 是由 T.A.贝蒂
M.H.惠勒 于 2018-06-06 设计创作，主要内容包括：公开了一种用于确定具有两个周边电极、远端电极和近端电极的生物传感器的故障安全值的方法和系统。将液体测量介质施加到生物传感器的毛细管通道。该方法包括向周边电极和近端电极施加交流电压,测量电导率以确定周边电极和近端电极之间的第一阻抗,向周边电极和远端电极施加交流电压,测量电导率以确定周边电极和远端电极之间的第二阻抗,使用第一阻抗和第二阻抗来确定一值,以及如果该值超出容差,则向用户提供错误消息。如果该值超出容差,则在反应区域中的电极和/或试剂中存在缺陷或断裂,并且该方法拒绝承认测试结果。(A method and system for determining a failsafe value for a biosensor having two peripheral electrodes, a distal electrode and a proximal electrode is disclosed. A liquid measurement medium is applied to a capillary channel of the biosensor. The method includes applying an alternating voltage to the peripheral electrode and the proximal electrode, measuring conductivity to determine a first impedance between the peripheral electrode and the proximal electrode, applying an alternating voltage to the peripheral electrode and the distal electrode, measuring conductivity to determine a second impedance between the peripheral electrode and the distal electrode, determining a value using the first impedance and the second impedance, and providing an error message to a user if the value is outside of a tolerance. If the value is outside of tolerance, there is a defect or break in the electrodes and/or reagents in the reaction region and the method refuses to acknowledge the test result.)
1. A method for error checking a biosensor, comprising:
applying a liquid measurement medium to a peripheral electrode, a proximal electrode and a distal electrode on the biosensor;
applying an alternating voltage to the peripheral electrode and the proximal electrode;
measuring a conductivity for determining a first impedance between the peripheral electrode and the proximal electrode;
applying the alternating voltage to the peripheral electrode and the distal electrode;
measuring a conductivity for determining a second impedance between the peripheral electrode and the distal electrode;
determining a value using the first impedance and the second impedance; and
if the value is outside of the tolerance, an error message is provided.
2. The method of claim 1, wherein the peripheral electrode is a sample sufficiency counter electrode.
3. The method of claim 1, wherein the peripheral electrode is a sample-sufficient working electrode.
4. The method of any one of claims 1 to 3, wherein the proximal electrode is one of a working electrode or a counter electrode and the distal electrode is the other of the working electrode or the counter electrode.
5. The method of any of claims 1 to 4, further comprising:
defects in the proximal electrode are detected.
6. The method of any of claims 1 to 5, further comprising:
defects in the distal electrode are detected.
7. The method of any of claims 1-6, wherein the value is a ratio formed between the first impedance and the second impedance.
8. The method of claim 7, wherein providing an error message occurs if the value is less than 1.0, the peripheral electrode is a sample-sufficient working electrode, the proximal electrode is a working electrode, and the distal electrode is a counter electrode.
9. The method of claim 7, wherein providing an error message occurs if the value is greater than 1.0, the peripheral electrode is a sample sufficiency counter electrode, the proximal electrode is a working electrode, and the distal electrode is a counter electrode.
10. The method of any one of claims 1 to 9, wherein the value is a ratio ZREAL(peripheral electrode-proximal electrode)/ZREAL(peripheral electrode-distal electrode), wherein the value less than 1.0 indicates that the distal electrode is defective.
11. The method of any one of claims 1 to 10, wherein the value is a ratio ZREAL(peripheral electrode-proximal electrode)/ZREAL(peripheral electrode-distal electrode), wherein the value greater than 1.0 indicates that the proximal electrode is defective.
12. The method of any of examples 1-11, further comprising:
applying an alternating voltage to the second peripheral electrode and the proximal electrode;
measuring a conductivity for determining a third impedance between the second peripheral electrode and the proximal electrode;
applying an alternating voltage to the second peripheral electrode and the distal electrode;
measuring a conductivity for determining a fourth impedance between the second peripheral electrode and the distal electrode;
determining a second value using the third impedance and the fourth impedance; and
if the second value is outside of the tolerance, a second error message is provided.
13. A measurement instrument for error checking a biosensor, the instrument comprising:
contacts of the first peripheral electrode, the second peripheral electrode, the proximal electrode, and the distal electrode electrically connected to the biosensor;
electronics that generate a test voltage and detect sensor signals from the first peripheral electrode, the second peripheral electrode, the proximal electrode, and the distal electrode;
a processor programmed to:
applying an alternating voltage to two electrodes of the biosensor and measuring a conductivity, wherein one electrode is a first peripheral electrode or a second peripheral electrode and the second electrode is a proximal electrode or a distal electrode, the conductivity being used to determine a first impedance between the two electrodes;
applying an alternating voltage to the remaining two electrodes of the biosensor and measuring the conductivity, which is used to determine a second impedance between the remaining two electrodes;
determining a value using the first impedance and the second impedance; and
providing an error message if the value is outside of the tolerance; and
an output unit providing an error message.
14. The instrument of claim 13, wherein the value is a ratio formed between the first impedance and the second impedance.
15. The instrument of any one of claims 13 to 14, wherein providing that the error message occurs if the value is less than 1.0, the first peripheral electrode is a sample-sufficient working electrode, the proximal electrode is a working electrode, and the distal electrode is a counter electrode.
16. The instrument of any one of claims 13 to 14, wherein providing that the error message occurs if the value is greater than 1.0, the second peripheral electrode is a sample sufficiency counter electrode, the proximal electrode is a working electrode, and the distal electrode is a counter electrode.
Technical Field
The present patent application relates generally to engineering and medical diagnostics, and more particularly, to electrochemical biosensors having multiple electrodes arranged to compare impedance measurements between the multiple electrodes to detect electrode breakage and/or reagent defects.
Background
Inexpensive disposable electrochemical biosensors typically employ one or two thin conductive layers formed on a flexible or semi-rigid substrate. The conductive layers are formed as electrodes, traces (trace) and contact pads on the biosensor, wherein the resistivity of the conductive traces connecting the reaction zone of the biosensor to the electronic circuitry in the test meter can measure hundreds of ohms or more. This resistance causes the potential along the length of the trace to drop such that the potential presented to the measurement electrode in the reaction zone is less than the potential applied by the tester to the contact pad of the biosensor in the contact zone of the biosensor. These substrates are susceptible to a variety of physical stresses that may stress or fracture the conductive traces. Stress can occur during manufacturing, shipping, handling by the user, or extreme storage conditions. The stress may be a series of cracks or localized failures, resulting in an unexpectedly high trace impedance. Severe stress may cause open circuits or fractures in one or more of the electrodes. Electrode breakage in the connecting trace can be detected by confirming a complete loop resistance or measuring an open circuit. Electrode breakage in the active reaction area can be difficult to detect and can adversely affect the normal operation of the biosensor, thereby introducing errors in the reported results.
The manufacturing process of the electrochemical biosensor may also include the continuous application of a thin layer of the reagent membrane, which may be susceptible to cracking after drying. In any of a variety of situations, a break in the reagent film may occur during the manufacturing process. For example, when mechanically cutting near or through the agent, a break may occur. As another example, cracking may occur due to physical stress such as twisting, bending, stretching or flexing of the base substrate during manufacturing. As yet another example, during the manufacturing process of the electrochemical biosensor, breakage may occur when the base substrate is compressed or clamped against debris or point defects on guide rollers.
In addition, reagent film rupture may also form over time, particularly after brief repeated or single prolonged exposure to high relative humidity. The dried reagent membrane is somewhat hydrophilic in design because it tends to absorb moisture. With increasing exposure time or relative humidity, the reagent film may partially hydrate and physically rearrange upon drying. The reconstituted reagent membrane may not be as homogeneous as desired and may be more prone to separation and/or rupture. The broken reagent film may extend into the underlying conductive trace and non-conductive support substrate, depending on the relative adhesion, elasticity and thickness. If severe, reagent rupture may lead to electrode breakage in the active reaction area of the biosensor. Electrode breakage can result in loss of function, including multiple harsh open circuits, or more subtle changes in the area of the effective or working electrode to counter electrode impedance, thus non-detectably and undesirably affecting accurate correction or calculation of the desired analyte concentration.
In certain manufacturing processes of electrochemical biosensors, the most common problem is the breakage of the external counter electrode, possibly due to intensified reagent rupture near the capillary inlet when cutting through the reagent and the flexible base. For example, where there are two counter electrode segments in the reaction zone, the counter electrode area is at least 1.5 times the working electrode area. Only external defects or breaks in the electrodes will have minimal effect on the DC response of the biosensor, which should be proportional to the area of the working electrode. Any defect in the integrity of the working electrode that affects its functional area will have a linear negative impact on the DC response and may inadvertently increase the working electrode to counter electrode impedance. A defective counter electrode segment may not adversely affect the DC response, but may result in the working electrode-to-counter electrode impedance appearing significantly higher than expected, resulting in overcorrection of the analyte concentration.
Accordingly, there is a need for improvements in this area.
Disclosure of Invention
A method of identifying deviations in a detection surface or reaction zone in a biosensor to reduce or eliminate the generation of false values is disclosed. The biosensor includes a reagent located within or adjacent to the capillary channel, and any suitable arrangement of electrode structures in the detection and reaction zones. These may include, but are not limited to, a working electrode, one or more counter electrodes, and one or more corresponding sample sufficiency (sample sufficiency) electrodes.
In operation, a low amplitude high frequency AC signal is applied between the peripheral electrode and the most proximal electrode, and a first impedance is measured. A similar AC signal is applied between the same peripheral electrode and the more distal electrode and a corresponding second impedance is measured. Due to their spatial relationship, the impedance between the peripheral electrode and the proximal electrode should be less than the impedance between the peripheral electrode and the distal electrode. Comparing the real parts of these two impedances can provide effective detection of proximal or distal electrode fracture defects under a wide range of test and material conditions. The electrode rupture detection function is enhanced by replicating the sequence using a second peripheral electrode.
Fail safe provides a method or means to identify biosensors having one or more damaged electrodes in the reaction area. By comparing the impedance between one electrode and its two nearest electrodes, a reasonable assessment of the integrity of the nearer electrode can be assessed. If the impedance between the base (peripheral) electrode and the most proximal electrode is higher than the impedance between the same base electrode and the more distal electrode, the proximal electrode is likely to be defective. The ratio of these resistances should be close to unity and substantially insensitive to normal variations in materials, manufacturing, environmental conditions or test solutions. The use of a low amplitude, high frequency AC signal minimizes polarization or interference with the potential of the electrochemical cell used to assess analyte concentration.
The method also includes providing a biosensor having an electrode-supporting substrate on which the first electrode is disposed. The first electrode includes a first body portion and a connective neck extending from the first body portion. The electrode-supporting substrate also has a second electrode disposed thereon, wherein the second electrode includes a second body portion and an opposing pair of connective necks. Each of the opposed pair of connecting necks extends from a respective end of the second body portion. In addition, at least two sample sufficiency electrodes are provided on the electrode-supporting substrate, each sample sufficiency electrode being positioned along a respective side edge of the electrode-supporting substrate, the sample sufficiency electrodes defining a gap therebetween. A spacer is also disposed on the electrode-supporting substrate, wherein the spacer includes at least one edge that defines a boundary of a capillary channel formed between the cover and the electrode-supporting substrate. In addition, at least two sample sufficiency electrodes surround the first electrode in the capillary channel, thereby forming a loop around the first electrode. The second body portion of the second electrode and the opposing pair of connective necks surround the first electrode in the capillary channel, forming a circuit around the first electrode. Alternative biosensors include other electrode patterns, including biosensors having three or four electrodes, in which failsafe can be determined.
Aspect 1 relates to a method for error checking a biosensor, the method comprising: applying a liquid measurement medium to a peripheral electrode, a proximal electrode and a distal electrode on the biosensor; applying an alternating voltage to the peripheral electrode and the proximal electrode, measuring a conductivity for determining a first impedance between the peripheral electrode and the proximal electrode, applying the alternating voltage to the peripheral electrode and the distal electrode, measuring a conductivity for determining a second impedance between the peripheral electrode and the distal electrode, determining a value using the first impedance and the second impedance, and providing an error message if the value is outside of a tolerance.
Aspect 2 relates to the method of aspect 1, wherein the peripheral electrode is a sample sufficiency counter electrode.
Aspect 3 relates to the method according to aspect 1, wherein the peripheral electrode is a sample sufficiency working electrode.
Aspect 4 relates to the method of any one of aspects 1-3, wherein the proximal electrode is one of the working electrode or the counter electrode and the distal electrode is the other of the working electrode or the counter electrode.
Aspect 5 relates to the method of any one of aspects 1-4, further comprising detecting a defect in the proximal electrode.
Aspect 6 relates to the method according to any one of aspects 1-5, further comprising detecting a defect in the distal electrode.
Aspect 7 relates to the method of any one of aspects 1-6, wherein the value is a ratio formed between the first impedance and the second impedance.
Aspect 8 relates to the method according to aspect 7, wherein if the value is less than 1.0, an error message is provided to occur, the peripheral electrode is a sample-sufficiency working electrode, the proximal electrode is a working electrode, and the distal electrode is a counter electrode.
Aspect 9 relates to the method according to aspect 7, wherein if the value is greater than 1.0, an error message is provided to occur, the peripheral electrode is the sample sufficiency counter electrode, the proximal electrode is the working electrode, and the distal electrode is the counter electrode.
Aspect 10 relates to the method of any one of aspects 1 to 9, wherein the value is the ratio ZREAL(peripheral electrode-proximal electrode)/ZREAL(peripheral electrode-distal electrode), wherein a value less than 1.0 indicates that the distal electrode is defective.
Aspect 11 relates to the method of any one of aspects 1 to 10, wherein the value is the ratio ZREAL(peripheral electrode-proximal electrode)/ZREAL(peripheral electrode-distal electrode), wherein the value greater than 1.0 indicates that the proximal electrode is defective.
Aspect 12 relates to the method of any one of aspects 1-9, further comprising applying an alternating voltage to the second peripheral electrode and the proximal electrode, measuring a conductivity for determining a third impedance between the second peripheral electrode and the proximal electrode, applying an alternating voltage to the second peripheral electrode and the distal electrode, measuring a conductivity for determining a fourth impedance between the second peripheral electrode and the distal electrode, determining a second value using the third impedance and the fourth impedance, and providing a second error message if the second value is outside of a tolerance.
Aspect 13 relates to a measurement instrument for error checking a biosensor, the instrument comprising: a first peripheral electrode electrically connected to the biosensor, a second peripheral electrode, contacts of the proximal electrode and the distal electrode, electronics to generate a test voltage and detect sensor signals from the first peripheral electrode, the second peripheral electrode, the proximal electrode and the distal electrode, a processor programmed to apply an alternating voltage to both electrodes of the biosensor and to measure a conductivity, wherein one electrode is the first peripheral electrode or the second peripheral electrode and the second electrode is the proximal electrode or the distal electrode, the conductivity being used to determine a first impedance between the two electrodes; applying an alternating voltage to the remaining two electrodes of the biosensor and measuring the conductivity, which is used to determine a second impedance between the remaining two electrodes; a value is determined using the first impedance and the second impedance and an error message is provided if the value is out of tolerance, and an output unit provides the error message.
Aspect 14 relates to the apparatus of aspect 13, wherein the value is a ratio formed between the first impedance and the second impedance.
Aspect 15 relates to the instrument of any one of aspects 13-14, wherein if the value is less than 1.0, providing an error message occurs, the first peripheral electrode is a sample-sufficient working electrode, the proximal electrode is a working electrode, and the distal electrode is a counter electrode.
Aspect 16 relates to the instrument of any one of aspects 13-14, wherein if the value is greater than 1.0, providing an error message occurs, the second peripheral electrode is a sample sufficiency counter electrode, the proximal electrode is a working electrode, and the distal electrode is a counter electrode.
Other forms, objects, features, aspects, benefits, advantages, and embodiments of the present invention will become apparent from the detailed description and drawings provided herein.
Drawings
FIG. 1 is a flow chart illustrating one example of a method of identifying deviations in the surface of a detection or reaction zone in a biosensor;
FIG. 2 is a perspective view of an exemplary biosensor;
FIG. 3 is a plan view of the biosensor shown in FIG. 2;
FIG. 4 is a plan view of a portion of the biosensor shown in FIG. 2, showing an exemplary electrode arrangement;
FIG. 5 is a plan view of a portion of the biosensor shown in FIG. 4, illustrating sample application;
FIG. 6 is a plan view of an analog switch matrix in a test meter or other device configured to use the biosensor shown in FIG. 2;
FIG. 7 is a plan view of a portion of the biosensor shown in FIG. 4, showing the impedance measurement from SSCE to the counter electrode and the impedance measurement from SSWE to the counter electrode being measured;
FIG. 8 is a plan view of a portion of the biosensor shown in FIG. 4 having a defect in the counter electrode, illustrating the impedance measurement from SSCE to the counter electrode and the impedance measurement from SSWE to the counter electrode being measured;
FIG. 9 plots | ZSSWE-CEAnd | Z! ZSSCE-CEThe test result of (1);
FIG. 10 plots | ZSSWE-WEAnd | ZSSCE-WEThe test result of (1);
FIG. 11 plots the test results from FIG. 9 with the x + -5 σ limit and the test results from the biosensor shown in FIG. 4 configured with various electrode breaks;
FIG. 12A is a plan view of a portion of the biosensor shown in FIG. 4 having a defect in the counter electrode;
FIG. 12B is a plan view of measuring defects in the counter electrode of FIG. 12A;
FIG. 12C is a graph representing different locations of defects in the counter and/or working electrodes of the biosensor shown in FIG. 4 to simulate the absence of 0%, 25%, 50%, 75%, or 95% of any of these electrodes;
FIG. 13 shows a one-sided comparison between a peripheral electrode and a proximal electrode for the biosensor of FIG. 2 and the same peripheral electrode and a similar but more distal secondary electrode;
FIG. 14A depicts a single side Z of the biosensor of FIG. 13REALA relationship;
FIG. 14B depicts a single side Z of the biosensor of FIG. 13REALA relationship;
FIG. 15 depicts a single side Z from FIG. 14AREALRelationship and impedance ratio of aqueous test solution and blood sample using about 2200 biosensors with nominal electrodes, and similar measurements using about 200 biosensors with intentionally defective electrodes;
FIG. 16 plots the one-sided Z from FIG. 14BREALA relationship and an impedance ratio of about 200 linearity and a sum blood sample of each of two intentional defects from a first test point (pilot);
FIG. 17 depicts a single side Z of a break in the sense electrode in the undamaged biosensor of FIG. 2 and the outer counter and/or working electrode of the biosensor of FIG. 2REALA ratio;
FIG. 18 depicts a single side Z of a break in the sense electrode in the undamaged biosensor of FIG. 2 and the outer counter and/or working electrodes of the biosensor of FIG. 2REALAnd (4) the ratio.
Detailed Description
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates. Although one embodiment of the present invention is shown in detail, it will be apparent to those skilled in the relevant art that some features that are not relevant to the present invention may not be shown for the sake of clarity.
The present application describes a method that enables identification of damaged electrodes in the reaction region of a biosensor, thereby preventing the generation of erroneous measurements. The measures taken to achieve this result are illustrated in fig. 1. The capillary channel of the biosensor contains a liquid measurement medium (at 100). Some examples of liquid measurement media include bodily fluids, such as blood, serum, plasma, saliva, aqueous environmental samples, treatment fluids, aqueous control fluids, or calibration fluids.
An alternating voltage is applied between the peripheral electrode and the proximal electrode at 110, and the alternating voltage (impedance) across the sample is measured at 120, giving a first impedance measurement. An alternating voltage is also applied between the perimeter electrode and the distal electrode at 130, and a second impedance measurement on the sample is measured at step 140. The real parts of these first and second impedances are determined and compared at 150. If the value from step 150 is outside of tolerance, an output error or a fail-safe error is provided to the user at 160. As discussed below, the tolerance can be any suitable value, such as a value greater than about 1.0, greater than about 1.043, greater than about 1.100, or greater. Values between 1.0 and about 1.1 may be considered nominal, while values less than about 1.0 or less than about 0.097 may also indicate failure.
By replicating this universal sequence for any of a plurality of different electrodes in a biosensor, the electrode rupture detection function can be enhanced. An alternating voltage is applied between the second peripheral electrode and the proximal electrode at 170, and the alternating voltage (impedance) across the sample is measured at 180, giving a third impedance measurement. At 190, an alternating voltage is applied between the second peripheral electrode and the distal electrode, and the impedance across the sample is measured at 200, producing a fourth impedance measurement. The real parts of the two impedances are determined and compared at 210. If the value from 210 is out of tolerance, an output error or a fail-safe error is provided to the user at 220. As discussed herein, the tolerance may be any suitable value, such as a value greater than 1.0 or less than 1.0, depending on which electrode is being tested for defects.
FIG. 1 illustrates one example of actions that may be taken on irregularities in biosensor electrodes. The disclosed impedance measurements and comparisons may be performed as shown in fig. 1 or in any other suitable order. For example, the impedance between the peripheral electrode and the distal electrode may be measured before the impedance between the peripheral electrode and the proximal electrode is measured. Similarly, the impedance between the second peripheral electrode and the distal electrode may be measured before the impedance between the second peripheral electrode and the proximal electrode is measured. Also, additional impedance between electrodes that may be present in a biosensor having additional electrodes may be measured. In other cases, the biosensor may have fewer electrodes, and thus some of the actions shown in FIG. 1 may be omitted accordingly.
A system for performing the method according to fig. 1 of the present application comprises a biosensor and a measuring instrument. The measuring instrument comprises at least one alternating voltage source and contacts for connection to electrodes in the biosensor. The measuring instrument further comprises control and measurement electronics to generate a voltage on the contacts and detect the sensor signals, and at least one processor to compare and correlate the sensor signals based on a program for performing the method according to the present application. The measuring instrument further comprises an output unit, such as a lamp, a light emitting diode, a display, a data interface, a printer connection, etc., for providing an error message if the value exceeds a tolerance. Software updates may be provided to the measurement instrument to fine tune tolerances and other aspects of the measurement process.
Fig. 2 shows a perspective view of an exemplary biosensor at 10. Fig. 2 is a plan view of the biosensor 10 shown in fig. 2. FIG. 3 is a plan view of a portion of the biosensor 10 shown in FIG. 2, illustrating an exemplary electrode arrangement. In an exemplary embodiment, the biosensor 10 includes an electrode-supporting substrate 12, an electrical conductor 14 formed on the electrode-supporting substrate 12 defining a plurality of electrode traces 16, 18, 19, 20, 21, and 22, a spacer 23 located on the electrode-supporting substrate 12, and a cover 24 located on the spacer 23. In some examples, electrical conductors 14 may form any number of electrode traces that enable biosensor 10 to function as described herein. However, in fig. 2 and 3, the spacer 23 is not shown for clarity.
As shown in fig. 1 and 2, the biosensor 10 may have a substantially rectangular shape; however, any of a variety of forms that enable biosensor 10 to function as described herein are also contemplated. In addition, the biosensor 10 may be any of a number of made from a roll of material, a sheet of material, or any other material feedstock in accordance with the principles of the present disclosure. In general, the material selection used to fabricate the biosensor 10 includes any material that is sufficiently flexible for roll processing, but is sufficiently rigid to provide a useful stiffness to the finished biosensor 10.
In an exemplary embodiment, the electrode-supporting substrate 12 of the biosensor 10 includes a first surface 42 facing the spacer 23 and a second surface 44 opposite the first surface 42. In addition, the electrode-supporting substrate 12 has opposing first and second ends 46, 48 and opposing side edges 50, 52 extending between the first and second ends 46, 48. In some examples, the first and second ends 46, 48 and the opposing side edges 50, 52 of the electrode-supporting substrate 12 form a generally rectangular shape. Alternatively, first and second ends 46, 48 and opposing side edges 50, 52 may be arranged to form any of a variety of shapes and sizes that enable biosensor 10 to function as described herein. In some examples, the electrode-support substrate 12 may be made of a flexible polymer including, but not limited to, polyester or polyimide, such as polyethylene naphthalate (PEN). Alternatively, the electrode-supporting substrate 12 can be made of any other suitable material that enables the electrode-supporting substrate 12 to function as described herein.
In an exemplary embodiment, the electrical conductors 14 forming the electrode traces 16, 18, 19, 20, 21, and 22 are provided on the first surface 42 of the electrode-supporting substrate 12. The electrical conductor 14 may be made of materials including, but not limited to, aluminum, carbon (e.g., graphite), cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium, mercury (e.g., amalgam), nickel, niobium, osmium, palladium, platinum, rhenium, rhodium, selenium, silicon (e.g., highly doped polysilicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium, and combinations thereof. In some examples, electrode traces 16, 18, 19, 20, 21, and 22 are isolated from the remainder of electrical conductor 14 by laser ablation or laser scribing, both of which are well known in the art. In this manner, electrode traces 16, 18, 19, 20, 21, and 22 may be fabricated by removing electrical conductor 14 extensively (such as by wide field ablation) or minimally (such as by scribing) from the area extending around the electrodes. Alternatively, the electrode traces 16, 18, 19, 20, 21, and 22 may be fabricated by other techniques, such as, for example, lamination, screen printing, photolithography, and the like.
In an exemplary embodiment, the biosensor 10 is a full width end dose ("FWED"; having a capillary channel bounded on one side) biosensor having a capillary channel 26 or inlet at the first end 46 of the electrode-supporting substrate. However, it is contemplated that the capillary passage 26 may also be a conventional capillary passage (i.e., bounded on more than one side). In the FWED biosensor, the spacer 23 extends between the opposite side edges 50, 52 of the electrode-supporting substrate 12 to partially form a capillary channel with the cover. It is envisaged that the spacer 23 may be made of a single component or even a plurality of components. Regardless, the spacer 23 should include an end edge 28 that is substantially parallel to and faces the first end 46 of the electrode-support substrate 12, thereby defining the boundary of the capillary channel 26 that extends across the entire width of the electrode-support substrate 12. Alternatively, and as described above, the end edge 28 may include portions between the first and second ends 46, 48 and the opposing side edges 50, 52 of the electrode-support substrate 12 to form a generally U-shaped pattern to define the boundary of the capillary channel 26 having a sample inlet (not shown) at the first end 46 of the biosensor 10. Other suitable embodiments contemplate that the end edge 28 forms a semi-oval, semi-circular, or other shaped capillary channel, and the one or more portions of the end edge 28 may include a linear or non-linear edge (not shown) along all or a portion of its length.
The spacer 23 is made of an insulating material such as, for example, a flexible polymer including adhesive-coated polyethylene terephthalate (PET) -polyester. One particular non-limiting example of a suitable material includes a white PET film coated on both sides with a pressure sensitive adhesive. The spacer 23 can be constructed of a variety of materials and includes an inner surface 25, which inner surface 25 can be coupled to the first surface 42 of the electrode-support substrate 12 using any one or combination of a variety of commercially available adhesives. In addition, when the first surface 42 of the support substrate 12 is exposed and not covered by the electrical conductor 14, the cover 24 may be coupled to support the electrode substrate 12 by welding, such as heating or ultrasonic welding. It is also contemplated that the first surface 42 of the electrode-support substrate 12 may be printed with product indicia or instructions (not shown), for example, for use with the biosensor 10.
Further, in the exemplary embodiment, cover 24 extends between opposite side edges 50, 52 of electrode-supporting substrate 12 and to first end 46 of electrode-supporting substrate 12. Alternatively, the cap 24 may extend a predetermined distance beyond the first end 46, which enables the biosensor 10 to function as described herein. In an exemplary embodiment, the capillary channel 26 is thus defined as the space between the cover 24 and the electrode support substrate 12 that is bounded by the first end 46 and the opposing side edges 50, 52 of the electrode support substrate 12 and the end edge 28 of the spacer 23.
The cover 24 may be made of an insulating material such as a flexible polymer including, for example, PET polyester. One particular non-limiting example of a suitable material includes a transparent or translucent PET film. The cover 24 may be constructed of various materials and include a lower surface 27, which lower surface 27 may be coupled to the spacer 23 using any one or combination of a variety of commercially available adhesives. In addition, the cover 24 may be coupled to the spacer 23 by welding (such as thermal welding or ultrasonic welding).
In an exemplary embodiment, biosensor 10 includes an outer counter electrode 30 and an inner counter electrode 32 extending across capillary channel 26 and coupled to electrode traces 18 and 19. In addition, biosensor 10 includes a working electrode 34, with working electrode 34 being located in capillary channel 26 between counter electrodes 30, 32. Working electrode 34 is coupled to traces 20 and 21. In addition, the biosensor 10 also includes a Sample Sufficiency Working Electrode (SSWE) 36 coupled to the electrode trace 22 and a Sample Sufficiency Counter Electrode (SSCE) 38 coupled to the electrode trace 16 located in the capillary channel 26. The SSWE36 and SSCE38 are located near the edge of the electrode-supporting substrate 12.
In the exemplary embodiment, SSCE 36 is coupled to contact pad SSE1 through electrode trace 22, and SSCE38 is coupled to contact pad SSE2 through electrode trace 16. Likewise, an outer counter electrode 30 and an inner counter electrode 32 are coupled to the electrode traces 18, 19. As shown in fig. 3, electrode trace 18 is coupled to contact pad CE, and electrode trace 19 is coupled to contact pads CS, B, and a. Further, working electrode 34 is coupled to electrode traces 20 and 21, with electrode trace 20 coupled to contact pad WE and electrode trace 21 coupled to contact pad WS. These contact pads provide conductive areas on the biosensor 10 to be contacted by the connector contacts of the test meter once the biosensor 10 is inserted into the test meter (not shown). It is further contemplated that the configuration of the electrodes, the number of electrodes, and the spacing between the electrodes may vary in accordance with the present disclosure. Thus, the biosensor 10 may include a greater or lesser number of electrodes than illustrated herein.
In the exemplary embodiment, working electrode 34 defines an effective working electrode area within capillary passage 26. The effective working electrode area is the area of the working electrode that is in contact with the fluid sample in the capillary channel 26 when the capillary channel 26 contains a sufficient volume of the fluid sample to initiate a measurement sequence. As seen in fig. 4, the working electrode 34 includes a body portion 60 extending laterally between the opposing side edges 50, 52 of the electrode-support substrate 12, and a connective neck 62 extending from the body portion 60 across the edge 28 of the capillary channel 26 (i.e., laterally from the body portion 60 toward the end 48 of the biosensor 10 opposite the capillary channel 26). The connection neck 62 is coupled to the electrode traces 20, 21 extending along one side of the electrode-supporting substrate 12. The spacer 23 is positioned so that the rim 28 extends across the connecting neck 62 and so that the body portion 60 is fully located within the capillary passage 26. Electrochemical detection reagents may be located on the working electrode 34, which provides an electrochemical probe for a particular analyte. The choice of a particular reagent depends on the analyte(s) to be measured, as is well known in the art. An example of a detection reagent that may be used in the biosensor 10 is a reagent for measuring glucose from a bodily fluid sample such as a whole blood sample.
In an exemplary embodiment, the inner counter electrode 32 and the outer counter electrode 30 are connected to electrode traces 18, 19 extending along one side of the electrode-supporting substrate 12. The outer counter electrode 30 extends laterally between the opposing side edges 50, 52 of the electrode-support substrate 12 and includes an extension trace 68 and a connecting neck 62, each of which extends from the body portion 70 across the edge 28 of the capillary channel (i.e., laterally from the body portion 70 toward the end 48 of the biosensor 10 opposite the capillary channel 26). In addition, the edges 28 of the capillary channel 26 extend along and partially overlap the inner counter electrode 32. In some examples, electrochemical detection reagents may be located on the inner counter electrode 32 and the outer counter electrode 30. As mentioned above, detection reagents provide electrochemical probes for specific analytes and are well known in the art, particularly for measuring glucose.
The biosensor 10 illustrates the effective electrode area with a reasonably symmetrical geometry in fig. 3 and 4. As illustrated in fig. 5, application of the sample is first detected by rapidly measuring the impedance between the external counter electrode 30 and the working electrode 34. Once the minimum conductivity is exceeded, sample sufficiency is then similarly determined by rapidly measuring the impedance between SSCE38 and SSWE 36. If the conductivity between the SSCE38 and the SSWE36 exceeds a programmable threshold within a programmable time out interval, the biosensor 10 is considered an acceptable dose (FIG. 5) and an analyte concentration measurement sequence can begin. If the minimum sample sufficient conductivity is not exceeded within the allowed time, an error will be indicated and the sequence aborted. SSCE38 and SSWE36 are primarily intended to ensure that the outer counter electrode 30 and working electrode 34 are sufficiently covered to reliably make analyte measurements.
The biosensor 10 in fig. 2-4 illustrates only one example of many possible arrangements of electrodes for electrochemical detection of a particular analyte. However, the principles discussed are applicable to any suitable geometry of electrodes in a biosensor. For example, the method may be applied to a biosensor having multiple working electrodes and a single counter electrode or any suitable configuration of proximal, distal and peripheral electrodes. Similarly, the disclosed methods may be effective for biosensors having any suitable arrangement of one or more counter electrodes, one or more working electrodes, and electrodes that perform similar functions as the disclosed SSCE and SSWE electrodes. No limitation should be implied based on the particular naming convention for the electrodes used in the disclosed examples. The terms "distal", "proximal", "working", and abbreviations such as "SSWE" and "SSCE" are exemplary and not limiting. Other biosensors and measurement devices may use different names for the various electrodes, but the principles disclosed herein still apply.
A tester or other device configured to use the biosensor 10 includes an analog switch matrix 80, which analog switch matrix 80 allows for programmable connection of single or multiple electrode contacts to a desired potentiostat function (fig. 6). The switch matrix 80, like the cross-point switches, allows reconfigurable connection of the excitation and response functions of the voltage regulator to a calibration load (RCAL) or any combination of up to seven biosensor contacts. One or more sensor contacts may be coupled together to add or extend the desired functionality. The inputs of the instrumentation amplifier select the positive and negative excitation feedback (sensing) inputs. These inputs can be selected from the biosensor contacts by closing the appropriate P and/or N switches, or by closing one or both of the vertical switches in fig. 6 as local feedback behind the switch array. Switch matrix 80 causes the potentiostat to interrogate external counter electrode 30 and working electrode 34 for remote excitation sensing detection through working sense and counter sense connections, and then connect to SSWE36 and SSCE 38. The switch matrix 80 allows for programmable selection of a plurality of alternative electrode connections for detecting accidental biosensor connections (shorts) or measuring other networks formed on the electrode-supporting substrate 12. A tester or other device configured to use the biosensor 10 is configured to apply a signal, such as, for example, an AC signal, to the biosensor 10 to check electrical continuity along the external counter electrode 30 and/or working electrode 34 prior to analyzing the biological fluid using the biosensor 10. Discontinuities along the outer pair of electrodes 30 and/or working electrode 34 result in an indication that biosensor 10 may be physically damaged. Thus, the tester may alert the user that the biosensor 10 has failed the integrity check and should therefore be discarded (i.e., the test results are fail-safe). If the biosensor 10 passes the integrity check (i.e., the tester confirms continuity along the outer counter electrode 30 and/or working electrode 34), the tester may make the user aware that the biosensor 10 may be safely used.
In a first failsafe, one method uses the spatial symmetry of the active electrode to detect a break in the outer counter electrode 30. In general, the impedance between the SSWE36 or SSCE38 and the external counter electrode 30 should be more affected by the external counter electrode 30 due to the proximity or proximity of the external counter electrode 30 to either of the SSWE36 and SSCE 38. Since both impedances are fairly affected by reagent flow rate, solution conductivity, environmental conditions, and metal resistivity, | Z | YSSWE-CEAndiz |)SSCE-CEShould track these situations. In one embodiment, the AC signal of amplitude is significantly less than the DC potential difference, which will even partially generate a glucose-dependent current, as described below. Applying a low-amplitude high-frequency AC signal between the external counter electrode 30 and a peripheral electrode such as SSWE36, and measuring the impedance as | ZSSWE-CEAbsolute value of (a). Next, a low-amplitude high-frequency AC signal is applied between the external counter electrode 30 and a peripheral electrode such as SSCE38, and the impedance is measured as | Z |SSCE-CEAbsolute value of (a). The impedance from SSCE38 to external counter electrode 30 is compared to the impedance from SSWE36 to external counter electrode 30. If none of the external counter electrodes 30 are presentA break or defect, the impedance between the outer counter electrode 30 and the peripheral electrode (e.g., SSWE 36) should be very similar or about equal to that between another peripheral electrode (e.g., SSCE 38) and the outer counter electrode 30 of equal area and spacing, as illustrated in fig. 7. If | Z |SSWE-CEAnd | Z |)SSCE-CEEqual or approximately equal, and the outer counter electrode 30, inner counter electrode 32, working electrode 34, reagent and sample appear symmetrically with respect to SSWE36 and SSCE38 in common, it can be assumed that the outer counter electrode 30 is intact. In other words, the external counter electrode 30 does not have any breakage or defect, and is not fail-safe.
Fig. 8 depicts an equivalent coupling to fig. 7, but illustrates the outer counter electrode 30 broken somewhere near the middle portion 31 of the outer counter electrode 30. The impedance from the SSCE38 in fig. 8 to the outer counter electrode 30 can be compared with similar measurements in fig. 7, but the impedance from the SSWE36 in fig. 8 to the outer counter electrode 30 will be higher, since now the SSWE36 is located further away from (a part of) the inner counter electrode 32. The effect will be even more pronounced if the counter electrode has only one outer counter electrode 30 and does not comprise an inner counter electrode 32. If | Z |SSWE-CEGreater than | Z | |)SSCE-CEThere is a high probability that the external counter electrode 30 is damaged or broken and an open circuit has been formed and provides fail-safe to the user.
In a second failsafe, one method uses the spatial symmetry of the active electrode to detect a break in working electrode 34. In one embodiment, an AC signal is applied that has a magnitude that is substantially less than, or even partially less than, the DC potential difference that would produce the glucose-dependent current, as described below. A low amplitude high frequency AC signal is applied between the working electrode 34 and a peripheral electrode such as SSWE36, and the impedance is measured as | Z |SSWE-WE. Next, a low-amplitude, high-frequency AC signal is applied between working electrode 34 and a peripheral electrode, such as SSCE38, and the impedance is measured as | ZSSCE-WE. The absolute values of the impedance from SSCE38 to working electrode 34 and the impedance from SSWE36 to working electrode 34 are compared. The side-to-side impedance between working electrode 34 and a peripheral electrode (e.g., SSWE 36) should be the same as working electrode 34 and haveAnother peripheral electrode (e.g., SSCE 38) of similar area and spacing is comparable. If | Z |SSWE-WEIs equal to or approximately equal to | ZSSCE-WEThen working electrode 34 may be assumed to be intact. In other words, working electrode 34 is free of any breakage or defect, and is not fail-safe. If | Z |SSCE-WEGreater than | Z | |)SSWE-WEIt is likely that working electrode 34 has been damaged or broken and an open circuit has been formed and provides fail-safe to the user.
FIG. 9 depicts | Z |. plotted along the y-axis as measured in ohms for a normal biosensor 10 that is assumed to be undamaged over a wide range of sample conductivities, alkali metal thicknesses, reagent film thicknesses, test point lifetimes, storage conditions, manufacturing variations, and test temperaturesSSWE-CEAnd | Z! ZSSCE-CEThe test results of (1). The impedance ratio was found to be approximately equal to 1 and in one form approximately 0.987.
FIG. 10 depicts | Z |. plotted along the y-axis as measured in ohms for a normal biosensor 10 that is assumed to be undamaged over a wide range of sample conductivities, alkali metal thicknesses, reagent film thicknesses, test point lifetimes, storage conditions, manufacturing variations, and test temperaturesSSWE-WEAnd | Z! ZSSCE-WEThe test results of (1). The impedance ratio is found to be approximately equal to 1 and in one form is approximately 0.973.
With respect to fig. 9 and 10, the near 1:1 relationship of the two sets of test results is quite noisy. FIG. 10 plots | Z | -Y charge for the same biosensor 10 tested with the x + -5 σ limit in FIG. 9SSWE-CEAndiz |)SSCE-CEThe ratio of (a) to (b). FIG. 10 also plots test results from the biosensor 10 configured with various electrode breaks illustrated in FIG. 12C, as described below. The goal of effective fail-safety would be to reliably identify all or nearly all of the damaged sensors that exceed these limits.
Test results
A first test run was performed with biosensors 10 each at either the working electrode 34 and/or the external counter electrode 30All containing different locations of intentional defects. Intentional defects include electrode breaks or gaps G in the working electrode 34 and/or the outer counter electrode 30, which are about 30 μm wide. A total of 15 different locations of intentional defects or electrode breaks in the electrode structure are illustrated in fig. 12A, 12B, and 12C. Design variations or electrode breaks were designed to simulate the 0%, 25%, 50%, 75% and 95% absence of working electrode 34 and/or outer counter electrode 30 illustrated in fig. 12C. Fig. 12A also includes an example (labeled volume 4) shown in an enlarged view. Ten variations of | ZSSWE-CEAndiz |)SSCE-CEAnd is labeled as volume 3, volume 4, volume 5, volume 6, volume 9, volume 10, volume 11, volume 15, volume 16, and volume 17. The test solutions include aqueous linearity solutions of various glucose concentrations at room temperature and nominal blood, and are used and applied to biosensor 10 labeled as volume 3, volume 4, volume 5, volume 6, volume 9, volume 10, volume 11, volume 15, volume 16, and volume 17. Summarized from FIG. 10, 82.3% of the test points | ZSSWE-CETo | Z |SSCE-CEIs within the x ± 5 σ limit or is not better than 17.7% of effective fail-safe.
The test shows that Z |SSWE-CEAndiz |)SSCE-CEThe two-sided comparison ratio of ratios of (a) does not provide adequate warning of irregularities in the equivalent coverage, contact resistance and electrode integrity for SSCE38 and SSWE 36. Therefore, as illustrated in fig. 13, the electrode breakage inspection is limited to single-sided comparison. This modified failsafe compares the impedance between the peripheral electrode (SSCE 38 or SSWE 36) and the proximal electrode (e.g., the primary electrode (outer counter electrode 30) (fig. 13)) and the same peripheral electrode and a similar but more distal secondary electrode (working electrode 34) (fig. 13). The "left" checks the challenge SSWE36 impedance and the "right" checks the challenge SSCE38 impedance. By design, the impedance between SSWE36 and the farther working electrode 34 should be greater than the impedance between SSWE36 and the closer (outer) counter electrode 30. The impedance between the SSWE36 and any undamaged secondary (external counter 30 or working 34) electrode is conducted by the sampleThe influence of the ratio, temperature, electrode area and sheet metal resistance is large.
However, these effects should be determined for a given sample sufficiency electrode (SSCE 38 or SSWE 36) regardless of the secondary electrode selected. ZREAL（SSWE-WE）/ZREALThe (SSWE-CE) distal/proximal impedance ratio should be unaffected by these factors and remain slightly greater than one over a wide range of sample and test conditions. Therefore, if Z isREAL（SSWE-WE）/ZREAL(SSWE-CE) distal/proximal impedance ratios of less than about 1.005 may be due to the outer counter electrode 30 being broken or defective and not extending uninterrupted as desired.
The right side check similarly queries SSCE 38. If the ratio Z isREAL（SSCE-WE）/ZREAL(SSCE-CE) between about 1.043 and about 1.100, it is likely due to the outer counter electrode 30 being broken or defective. If Z isREAL（SSCE-WE）/ZREAL(SSCE-CE) distal/proximal impedance ratio greater than about 1.100, likely due to large ZREAL(SSCE-WE) impedance, which indicates that working electrode 34 is broken or defective. FIGS. 14A and 14B show a single side Z for a nominal biosensor 10REALThe relationship is extremely predictable in biosensor lifetime, reagent thickness, manufacturing limits, metallization thickness, test environment, and solution type and conductivity (HCT).
To determine defect detectability, the impedance data of fig. 14A and 14B for approximately 2,200 nominal biosensors 10 are plotted as an impedance ratio, labeled a in fig. 15 and B in fig. 16. Figures 15 and 16 include the nominal mean x ± 5 σ limits for each single-sided ratio. It has been found that, although not apparent in fig. 17A and 17B, the change in the SSCE-side ratio (fig. 16) is smaller than the SSWE-side ratio (fig. 15). Adjacent to the variation of the normal biosensor 10 plotted in fig. 15 and 16, a single-sided SSWE and SSCE impedance ratio of 200 linearity is plotted along with two intentionally defective blood samples from the test coupons: a 50% roll 17 lacking the working electrode 34 and a 25% roll 5 lacking the outer counter electrode 30. It is found from fig. 15 that the single-sided SSWE impedance ratio cannot reliably distinguish the midpoint break in the working electrode 34 of the test coupon 17 from the nominal material, but easily detects a 25% broken counter electrode of the test coupon 5. In other words, the single-sided SSWE impedance ratio was less than 1 in all test results, indicating the presence of an electrode break or defect in the outer counter electrode 30. It is found from fig. 16 that the single-sided SSCE impedance ratio is not effective in distinguishing a break in the outer counter electrode 30 of the roll 5 from the nominal material, but reliably detects each instance of a midpoint break in the working electrode 34 of the test coupon roll 17. In other words, the single-sided SSCE impedance ratio is greater than 1 in all test results, indicating the presence of an electrode break or defect in the working electrode 34.
FIGS. 17 and 13 show a single side Z for linearityREALThe ratio and blood sample on undamaged biosensor 10, with electrode breakage caused by all test spots in rolls 3, 4, 5, 6, 9, 10, 11, 15, 16 and 17, as discussed previously. FIG. 17 plots the ratio ZREAL（SSWE-WE）/ZREAL(SSWE-CE), which ratio is intended to detect a fracture in the outer counter electrode 30. This single-sided ratio reliably detects a defect for each outer counter electrode 30, but cannot distinguish a roll 17 with a complete outer counter electrode 30 and a midpoint break in the working electrode 34 in the biosensor 10 from a normal biosensor 10 without any electrode breaks. FIG. 13 is a graph of the rate Z of detecting a break or defect in the working electrode 34REAL（SSCE-WE）/ZREAL(SSCE-CE). The ratio ZREAL（SSCE-WE）/ZREAL(SSCE-CE) reliably detects the ratio Z in FIG. 17 in the working electrode 34 of the roll 17REAL（SSWE-WE）/ZREAL(SSWE-CE) "missed" midpoint breaks. Ratio ZREAL（SSCE-WE）/ZREAL(SSCE-CE) the majority of the remaining breaks in working electrodes 34 of the evaluated rolls 3, 4, 5, 6, 9, 10, 11, 15, 16 and 17 were known, but the complete working electrodes 34 from rolls 3, 4 and 5 could not be distinguished from the normal biosensor 10. Ratio ZREAL（SSCE-WE）/ZREAL(SSCE-CE) in distinguishing between a midpoint break in the roll 15 (working electrode 34) and the presence of an external counter electrode 30 on the biosensor 105%) is also not effective with a normal biosensor 10 without any electrode breakage, due to the high Z generated by the small stub (stub) of the external counter electrode 30REAL(SSCE-CE) to counteract the increased Z due to the break in the center of the working electrode 34REAL(SSCE-WE). Ratio ZREAL（SSWE-WE）/ZREAL(SSWE-CE) easily identifies the volume 15 as defective. Ratio Z for a complete working electrode 34 of roll 9REAL（SSCE-WE）/ZREAL(SSCE-CE) detection rule fails. Ratio Z for volume 9REAL（SSCE-WE）/ZREAL(SSCE-CE) is less than 1 because of the molecule ZREAL(SSCE-WE) is relatively normal, but the denominator ZREAL(SSCE-CE) is much larger than expected due to the largely unconnected external counter electrode 30. Ratio ZREAL（SSWE-WE）/ZREAL(SSWE-CE) and ratio ZREAL（SSCE-WE）/ZREAL(SSCE-CE) is insensitive to variations in glucose concentration, sample type, hematocrit, temperature, reagent flow rate, capillary height, spacer placement, metallization limits, biosensors stored in closed vials for more than two years, use case exposure, raw materials, manufacturing processes, environmental conditions, and test solutions.
Exemplary embodiments of electrode arrangements for biosensors are described above in detail. The apparatus and methods are not limited to the specific embodiments described herein, but rather, operations of the methods and components of the systems may be utilized independently and separately from other operations or components described herein. For example, the methods and devices described herein may have other industrial or consumer applications and are not limited to practice with the biosensor assemblies described herein. Rather, one or more embodiments may be implemented and utilized in connection with other industries.
While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes, equivalents, and modifications that come within the spirit of the invention as defined by the following claims are desired to be protected. All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and set forth in its entirety herein.
