Reference electrode for potential measurement system

Abstract

The invention provides a reference electrode for a potentiometric measurement system for measuring an analyte in a body fluid sample, comprising an Ag / AgCl conductor element (8) in contact with a reference electrolyte (7) wherein the chloride ion (Cl-) concentration in the reference electrolyte (7) is between 50 mmol / L and 200 mmol / L, and wherein the sodium ion (Na +) concentration in the reference electrolyte (7) is preferably between 70 mmol / L and 270 mmol / L, and wherein the Ag / AgCl conductor element (8) is in contact with the reference electrolyte (7). And wherein the ionic strength of the reference electrolyte (7) is between 75 mmol / L and 300 mmol / L. The invention also provides a potentiometric measurement system and a method for potentiometric measurement of an analyte in a body fluid sample.

Description

TECHNICAL FIELD

[0001] The present invention relates to a reference electrode and to a potentiometric system for measuring an analyte in a sample of a body fluid. BACKGROUND

[0002] In the field of clinical analysis, potentiometric systems are often used to determine the concentration of various ions, such as Na + , K + , Ca 2+ and Cl - , in a sample of a body fluid, such as a blood sample. These systems typically comprise one or more ion selective electrodes (ISE) and a reference electrode. The ISE is designed to respond selectively to a specific ion in the sample, providing a potential proportional to the activity of that ion. However, since the individual potentials cannot be measured, a reference electrode is required for the potentiometric measurement of ion activity using ISEs. The reference electrode acts as a stable half-cell, providing a constant potential against which the potential of the ISE can be measured. This two-electrode system, consisting of an ISE and a reference electrode, enables the determination of the ion activity in the sample, and hence the ion concentration.

[0003] One of the most common forms of reference electrode uses an Ag / AgCl conductor element in contact with a reference electrolyte, typically a high concentration or saturated KCl. The role of the reference electrolyte is to maintain a stable ionic environment around the Ag / AgCl conductor element, which is crucial for the stability and reproducibility of the reference electrode potential. However, the reference electrolyte is also in contact with the sample fluid to allow the potential of the sample to be measured against the stable potential of the reference electrode. Typically, this contact is provided through a diffusion barrier, such as an ion-permeable membrane or a porous plug, to control the interaction between the internal reference electrolyte and the external sample solution. As a result, ions can diffuse from the reference electrolyte into the sample and vice versa.

[0004] The exchange of electrolytes between the reference electrolyte and the sample presents several challenges. Firstly, the half-cell potential of the Ag / AgCl element depends on the chloride ion concentration at the electrode conductor element. Therefore, the continuous leaching of chloride ions from the reference electrolyte into the sample causes the reference potential to drift continuously. This manifests as a signal drift, making the measurement less reliable over time. Secondly, since different ions have different mobilities in the diffusion barrier, a diffusion potential, known as the liquid junction potential, can arise, which can interfere with the measured potential.

[0005] Typically, these challenges are addressed by providing a saturated or high concentration of KCl in the reference electrolyte. This makes the K + and Cl -Ions can continuously cross the diffusion barrier from the reference electrolyte, thus flooding the diffusion of other ions, reducing the difference in diffusion rates, and consequently reducing the liquid junction potential. However, the continuous leaching of KCl into the sample causes a sustained decrease in the concentration of the reference electrolyte, resulting in signal drift. This typically requires periodic refilling of the reference electrolyte to replenish KCl. Another issue is that when high concentrations of KCl are present... + and Cl - When ions leach into the sample channel, they can distort the signal obtained from the ISE located there. Therefore, the sample channel typically needs to be flushed frequently to wash away excess ions from the reference electrolyte.

[0006] US 2008 / 0149482 A1 relates to a reference electrode and a reference solution for the reference electrode. The reference solution comprises 1 M to saturated aqueous KCl solution; 0.6% to 2.46% (by weight of solution) of a secondary water-soluble inorganic chloride salt other than KCl; and 4.6% to 15.93% (by weight of solution) of an evaporation inhibitor.

[0007] WO 2022 / 245347 A1 discloses a solid-state reference electrode based on a polymer film. A concentrated supporting electrolyte (such as lithium acetate with a concentration in the range of 3 M to 5 M) is used to reduce the liquid junction potential.

[0008] EP 3 729 067 B1 discloses a reference electrode comprising an Ag / AgCl element located in an expandable polymer matrix filled with an electrolyte salt. The electrolyte salt (such as KCl and / or NaCl) is preferably present in microcrystalline form and a saturated aqueous solution, such that when dissolved KCl leaches out, it is replenished by more KCl entering the solution.

[0009] Further reference electrodes are disclosed in, for example, US 4,706,678 A, JPH 01118762 A, US 2020 / 319132A1, US 2001 / 025790 A1 and US 2016 / 033438 A1.

[0010] A common drawback of the reference electrodes described in the prior art is the continuous leaching of the reference electrolyte into the sample. This leads to problems such as reduced reference potential stability, signal drift, interference with ISEs, high maintenance costs due to the need for frequent flushing of the sample channel and / or replenishment of the reference electrolyte, and shortened reference electrode lifespan. Therefore, novel and improved reference electrodes and potential measurement systems are needed to address at least some of these drawbacks. The object of this invention is to provide such a reference electrode and measurement system. Summary of the Invention

[0011] Therefore, the present invention provides a reference electrode for a potential measurement system for measuring analytes in body fluid samples. This reference electrode includes an Ag / AgCl conductor element in contact with a reference electrolyte, wherein chloride ions (Cl-) in the reference electrolyte... - The concentration of sodium ions (Na) in the reference electrolyte (7) is between 50 mmol / L and 200 mmol / L. + The concentration is preferably between 70 mmol / L and 270 mmol / L, and the ionic strength of the reference electrolyte is between 75 mmol / L and 300 mmol / L.

[0012] In another aspect, the present invention provides a potential measurement system for measuring the potential of an analyte in a body fluid sample, the potential measurement system comprising a reference electrode according to the present invention and at least one ion-selective electrode (ISE).

[0013] In another aspect, the present invention provides a method for potentiometric measurement of analytes in body fluid samples using a potentiometric measurement system according to the present invention, the method comprising the following steps:

[0014] - Contact the at least one ISE with a body fluid sample;

[0015] - Obtain a potential reading from the at least one ISE; and

[0016] - Determine the estimated ion activity based on the potential reading.

[0017] As mentioned above, prior to this invention, the use of high-concentration reference electrolytes in reference electrodes was firmly established. During the development of this invention, it was unexpectedly discovered that a more stable and durable reference electrode can be provided by employing a lower reference electrolyte concentration. Specifically, it was found that when the chloride ion concentration of the reference electrolyte is relatively low, for example, 50 mmol / L to 200 mmol / L, the reference potential is more stable over a longer period. According to the inventors, this is because bodily fluid samples, particularly blood samples, typically have chloride ion concentrations in this range, typically 97 mmol / L to 105 mmol / L. When the chloride ion concentration of the reference electrolyte is comparable to that in the sample, the chloride ion concentration gradient is smaller, and therefore the change in chloride ion concentration in the reference electrolyte is smaller when the reference electrolyte comes into contact with the sample. Since the reference potential measured using an Ag / AgCl conductor element depends on the chloride ion concentration, this results in a more stable signal. While chloride ions have the greatest impact on Ag / AgCl conductor elements, similar considerations apply to the concentrations of other ions contained in the reference electrolyte. Typically, the ionic strength of bodily fluid samples (such as blood samples) is approximately 150 mmol / L. When the ionic strength of the reference electrolyte is within a similar range, such as between 75 mmol / L and 300 mmol / L, this reduces the change in ion concentration over time, thereby making the electrolyte composition more stable.

[0018] Furthermore, when using a high-concentration reference electrolyte, the contact between the reference electrolyte and the sample not only causes changes in the concentration of the reference electrolyte but also in the concentration of the sample. For example, when the reference electrolyte contains a high concentration of KCl, there is a K... + and Cl - Ions flow into the sample. Therefore, the K in the sample... + and Cl - The concentration will increase, which can interfere with the ISE used to measure these ions. Therefore, frequent rinsing of the sample channel is usually necessary to wash away excess ions from the reference electrolyte. This problem can be largely avoided by using physiological or near-physiological ion concentrations in the reference electrolyte.

[0019] When a reference electrolyte comes into contact with the sample being tested, a diffusion potential, known as the liquid junction potential, is generated at the interface. This is due to the different rates of interdiffusion of ions in the two solutions. In conventional reference electrodes, this problem is solved by providing a saturated or high concentration of KCl in the reference electrolyte. This allows KCl to be absorbed into the electrolyte. + and Cl - Ions can continuously flow from the reference electrolyte into the sample, thereby swamping the diffusion of other ions, reducing the difference in diffusion rates, and thus lowering the liquid junction potential. Because K... + and Cl -Since the ions have similar mobilities, the liquid junction potential is minimized. The reference electrode in this invention employs a different strategy to minimize the liquid junction potential. By ensuring that the reference electrolyte and the sample have similar ionic strengths and similar electrolyte compositions, a large liquid junction potential is prevented from the outset.

[0020] Furthermore, the inventors have developed a strategy to further reduce the influence of liquid junction potential. More specifically, when using a reference electrode with one or more ISEs in a potential measurement system, they found that the ISE can be used to estimate the ion activity in the sample, and then this estimated ion activity can be used to estimate the diffusion potential between the sample and the reference electrolyte. This estimated diffusion potential can then be used to correct the previously determined ion activity to obtain a corrected ion activity. Furthermore, these corrected ion activities can be used to provide even more accurate diffusion potential estimates, i.e., second-order estimated diffusion potentials, and these second-order estimated diffusion potentials can be used to obtain even more accurate second-order corrected ion activities. In this way, the negative impact of liquid junction potential can be minimized without the need for high concentrations of KCl in the reference electrolyte.

[0021] In the reference electrode of the present invention, the Ag / AgCl conductor element is in contact with the reference electrolyte, preferably immersed in the reference electrolyte. Preferably, the reference electrolyte is an aqueous electrolyte.

[0022] As mentioned above, it is advantageous if the ionic strength of the reference electrolyte is similar to that of a typical bodily fluid sample (especially a blood sample). Therefore, preferably, the ionic strength of the reference electrolyte is between 80 mmol / L and 280 mmol / L, more preferably between 85 mmol / L and 270 mmol / L, more preferably between 90 mmol / L and 260 mmol / L, more preferably between 95 mmol / L and 250 mmol / L, more preferably between 100 mmol / L and 240 mmol / L, more preferably between 105 mmol / L and 230 mmol / L, more preferably between 110 mmol / L and 220 mmol / L, more preferably between 115 mmol / L and 210 mmol / L, more preferably between 120 mmol / L and 200 mmol / L, more preferably between 125 mmol / L and 190 mmol / L, more preferably between 130 mmol / L and 180 mmol / L, more preferably between 135 mmol / L and 170 mmol / L, and more preferably between 140 mmol / L and 160 mmol / L. Between mmol / L.

[0023] Normally, chloride ions (Cl) in the blood -The concentration of Cl in the reference electrolyte is between 97 mmol / L and 105 mmol / L. Therefore, if the Cl concentration in the reference electrolyte... - The concentration is preferably between 55 mmol / L and 190 mmol / L, more preferably between 60 mmol / L and 180 mmol / L, more preferably between 65 mmol / L and 170 mmol / L, more preferably between 70 mmol / L and 160 mmol / L, more preferably between 75 mmol / L and 150 mmol / L, more preferably between 80 mmol / L and 140 mmol / L, more preferably between 85 mmol / L and 130 mmol / L, more preferably between 90 mmol / L and 120 mmol / L, more preferably between 95 mmol / L and 110 mmol / L, and more preferably between 97 mmol / L and 105 mmol / L.

[0024] To further minimize the change in the composition of the reference electrolyte over time and to minimize the liquid junction potential, it is preferable that the reference electrolyte has an electrolyte concentration similar to that of a typical blood sample. Typically, blood contains sodium ions (Na₂O₃). + The concentration of Na+ is between 140 mmol / L and 148 mmol / L. Therefore, if the reference electrolyte contains Na+... + The concentration is preferably between 70 mmol / L and 270 mmol / L, more preferably between 80 mmol / L and 250 mmol / L, more preferably between 90 mmol / L and 230 mmol / L, more preferably between 100 mmol / L and 210 mmol / L, more preferably between 110 mmol / L and 190 mmol / L, more preferably between 120 mmol / L and 170 mmol / L, more preferably between 135 mmol / L and 155 mmol / L, and more preferably between 140 mmol / L and 148 mmol / L.

[0025] Normally, potassium ions (K) in the blood + The concentration of K in the reference electrolyte is between 3.7 mmol / L and 5.1 mmol / L. Therefore, if the K concentration in the reference electrolyte is... +The concentration is preferably between 2.1 mmol / L and 8.3 mmol / L, more preferably between 2.3 mmol / L and 7.9 mmol / L, more preferably between 2.5 mmol / L and 7.5 mmol / L, more preferably between 2.7 mmol / L and 7.1 mmol / L, more preferably between 2.9 mmol / L and 6.7 mmol / L, more preferably between 3.1 mmol / L and 6.3 mmol / L, more preferably between 3.3 mmol / L and 5.9 mmol / L, more preferably between 3.5 mmol / L and 5.5 mmol / L, and more preferably between 3.7 mmol / L and 5.1 mmol / L.

[0026] Normally, calcium ions (Ca) in the blood 2+ The concentration of Ca in the reference electrolyte is between 1.08 mmol / L and 1.30 mmol / L. Therefore, if the Ca concentration in the reference electrolyte... 2+ The concentration is preferably between 0.5 mmol / L and 2.5 mmol / L, more preferably between 0.6 mmol / L and 2.3 mmol / L, more preferably between 0.7 mmol / L and 2.1 mmol / L, more preferably between 0.8 mmol / L and 1.9 mmol / L, more preferably between 0.9 mmol / L and 1.7 mmol / L, more preferably between 1.0 mmol / L and 1.5 mmol / L, and more preferably between 1.08 mmol / L and 1.30 mmol / L.

[0027] Typically, the pH of blood is between 7.35 and 7.45. Therefore, it is preferred that the pH of the reference electrolyte is between 6.5 and 8.5, more preferably between 6.6 and 8.3, more preferably between 6.7 and 8.1, more preferably between 6.8 and 8.0, more preferably between 6.9 and 7.9, more preferably between 7.0 and 7.8, more preferably between 7.1 and 7.7, more preferably between 7.2 and 7.6, more preferably between 7.3 and 7.5, and most preferably between 7.35 and 7.45.

[0028] In the case of this invention, it has proven advantageous if the reference electrolyte is at least partially immobilized. In this case, partial immobilization may imply increased ion mobility (preferably Cl). - The ion mobility is reduced compared to that in water. This slows diffusion and reduces ion mobility between the reference electrolyte and the sample, thus further improving the stability and durability of the reference electrode.

[0029] In a preferred embodiment, the reference electrolyte is in gel form. Preferably, the reference electrolyte contains a gel-forming agent, preferably a polysaccharide, more preferably agarose. A concentration of the gel-forming agent in the reference electrolyte between 0.2 wt.% and 20 wt.% is particularly preferred, more preferably between 0.5 wt.% and 10 wt.%, and more preferably between 1 wt.% and 4 wt.%. This reference electrolyte is highly suitable for reducing ion mobility.

[0030] In another preferred embodiment, the reference electrolyte contains a humectant. Preferably, the reference electrolyte contains a polyol, preferably selected from carbohydrates, sugar alcohols, glycerol, and / or ethylene glycol; especially glycerol. Preferably, the concentration of the polyol in the reference electrolyte is between 30 wt.% and 80 wt.%, more preferably between 40 wt.% and 70 wt.%. Providing such a humectant advantageously reduces the drying of the reference electrolyte, especially when the reference electrolyte is a gel. This increases its service life and allows the reference electrode to be stored for a longer period of time.

[0031] In yet another preferred embodiment, the reference electrolyte comprises a porous additive, preferably fumed silica. The addition of this porous additive is particularly advantageous when the reference electrolyte is a gel, as it reduces shrinkage of the gel during drying.

[0032] Preferably, the reference electrolyte contains 10 wt.% to 90 wt.% water, more preferably 15 wt.% to 80 wt.% water, more preferably 20 wt.% to 60 wt.% water, and even more preferably 25 wt.% to 40 wt.% water.

[0033] The potential measurement system according to the invention includes a reference electrode according to the invention and at least one ion-selective electrode (ISE). As described above, including at least one ISE allows for at least partial correction of the diffusion potential, since the signal from the ISE can be used to estimate the corresponding ion activity, thereby estimating the diffusion potential generated by said ion.

[0034] For example, the diffusion potential can be estimated using Henderson's diffusion potential equation as follows:

[0035]

[0036] in:

[0037] ED = Electrochemical diffusion potential

[0038] = the transport number of ion i

[0039] = Charge number of ion i

[0040] = Activity of ion i in phase 1 (in the sample)

[0041] = Activity of ion i in phase 2 (in reference electrolyte)

[0042] The transport number of each ion is related to the conductivity data and the diffusion coefficient. An optimization method can be used to estimate the applied diffusion coefficient to minimize the residual diffusion potential.

[0043] Preferably, the at least one ISE is selected from the group consisting of: chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+ Selective electrodes and / or potassium ions (K) + Selective electrode.

[0044] During the research process of this invention, it has been proven that Cl - The effect of ions on diffusion potential is generally the greatest among all ions (see Example 2; comparison of Model B and Model C). Therefore, for ions composed of Cl... - Correcting for diffusion potential caused by ions can significantly improve measurement accuracy. Therefore, in a preferred embodiment, the at least one ISE is a chloride ion (Cl... - Selective electrode.

[0045] In another preferred embodiment, the at least one ISE is a sodium ion (Na+). + Selective electrode. It has been proven that Na... + Ions typically have a particularly large influence on diffusion potential. When measuring Cl... - and Na + At this point, the diffusion potential can be estimated relatively accurately, thus significantly improving measurement accuracy (see Example 2 and Model C in Figure 2). Therefore, if the potential measurement system includes at least chloride ions (Cl... - ) and sodium ions (Na) + Selective electrodes are particularly preferred.

[0046] When more ions are measured using an ISE, this allows for a more accurate estimation of the diffusion potential, and thus a more accurate determination of the ion concentration. Therefore, in a preferred embodiment, the potential measurement system includes at least two, preferably at least three, ISEs selected from the group consisting of chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+Selective electrodes and / or potassium ions (K) + Selective electrode. In a preferred embodiment, the potential measurement system includes at least a chloride ion (Cl) selective electrode. - Selective electrode and sodium ions (Na) + Selective electrode. In another preferred embodiment, the potential measurement system includes at least a chloride ion (Cl) electrode. - Selective electrode, sodium ions (Na) + Selective electrodes and calcium ions (Ca) 2+ Selective electrode. In another preferred embodiment, the potential measurement system includes at least a chloride ion (Cl) selective electrode. - Selective electrode, sodium ions (Na) + Selective electrode and potassium ion (K) + Selective electrode.

[0047] When the potential measurement system contains at least chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+ Selective electrode and potassium ion (K) + This is particularly preferred when using a selective electrode. When measuring all four ions, it is possible to estimate not only the diffusion potential caused by these four ions, but also the diffusion potential caused by the remaining anions, for which ISE may not be readily available (e.g., HCO3-). - Therefore, the hypothetical ion pairs can be determined as follows:

[0048] - From K + The signal from the selective electrode can be used to estimate the concentration of the salt KCl (because Cl... - (The main anions present in blood samples).

[0049] - From Ca 2+ The signal from the selective electrode can be used to estimate the concentration of the salt CaCl2 (because Cl... - (The main anions present in blood samples)

[0050] - From Cl - The signal from the selective electrode can be used to estimate the NaCl concentration (based on [NaCl] = [Cl-]). - (Estimation of {[KCl+ CaCl2]})

[0051] Since the total charge of a blood sample should be neutral, and since blood samples should not contain high concentrations of any other cations, they can be mixed with any remaining anions (such as HCO3-) in the sample. -The residual anion concentration corresponding to the concentration of Na+ is estimated as follows: [anion] = [Na+] + ] – [NaCl].

[0052] For the residual anion concentration, an optimization method can be used to estimate the effective transport number in order to minimize the residual diffusion potential, thus taking Cl into account when estimating the diffusion potential. - Na + Ca 2+ K + And residual anions. This makes the measurements particularly accurate (see Example 2 and Model E in Figure 2).

[0053] In a preferred embodiment, the potential measurement system includes a processor configured to perform the following steps:

[0054] A. Obtain a potential reading from at least one ISE;

[0055] B. Determine the estimated ion activity based on the stated potential reading;

[0056] C. Determine the estimated diffusion potential based on the estimated ion activity; and

[0057] D. Correct the estimated ion activity based on the estimated diffusion potential to obtain the corrected ion activity.

[0058] The estimated ion activity determined in step B is the original ion activity before correcting for the diffusion potential. The estimated ion activity corresponds to the activity of the ion selected by the ISE; for example, when the ISE is Cl... - In the case of selective electrodes, step B determines the initial Cl. - Activity. The raw ion activity obtained from step B can then be used in step C, for example, to estimate the diffusion potential induced by the ion using the Henderson equation described above. The estimated diffusion potential can then be used to correct the potential reading obtained in step A to determine the corrected potential, thereby determining the corrected ion activity.

[0059] As described above, it is advantageous to use multiple ISEs so that the diffusion potential can be estimated using multiple measurements of the original ion activity. Therefore, in a preferred embodiment, the potential measurement system includes at least two ISEs, preferably at least three ISEs, more preferably at least four ISEs, and the processor is configured to perform the following steps:

[0060] A. Obtain potential readings from each ISE;

[0061] B. Determine the estimated ion activity based on each potential reading;

[0062] C. Determine the estimated diffusion potential based on the estimated ion activity; and

[0063] D. Correct the estimated ion activity based on the estimated diffusion potential to obtain the corrected ion activity.

[0064] For example, when the potential measurement system includes at least Cl - Selective electrode and Na + When using a selective electrode, Cl can be determined in step B. - and Na + The original activity. In step C, it can be based on the Cl in the sample. - and Na + The activity is used to estimate the diffusion potential, and the estimated diffusion potential can be used to correct the potential reading obtained in step A, thereby obtaining the corrected Cl. - and Na + activity.

[0065] If the potential measurement system includes at least Cl - Selective electrode, Na + Selective electrode, Ca 2+ Selective electrode and K + A selective electrode is particularly preferred. In this case, the estimated Cl can be determined in step B. - Na + Ca 2+ and K + Activity. Based on these activities, the residual anion activity can be determined as described above. Then, based on the estimated Cl... - Na + Ca 2+ K + The estimated diffusion potential in step C, along with the residual anion activity determination, can lead to particularly accurate corrections in step D.

[0066] Once the corrected ion activities are obtained in step D, these corrected ion activities can be used to estimate the diffusion potential more accurately. Therefore, in a preferred embodiment, the processor is configured to further perform the following steps:

[0067] E. Determining the second-order estimated diffusion potential based on corrected ion activity; and

[0068] F. The corrected ion activity is further corrected based on the second-order estimated diffusion potential to obtain the second-order corrected ion activity.

[0069] In a particularly preferred embodiment, steps E and F are repeated during the iteration process. Therefore, based on the second-order corrected ion activity, the third-order estimated diffusion potential can be estimated again using the second-order corrected ion activity, and then the third-order estimated diffusion potential can be used to obtain the third-order corrected ion activity, and so on.

[0070] The method of the present invention for measuring analytes in body fluid samples using the potential measurement system of the present invention includes the following steps:

[0071] - Contact the at least one ISE with a body fluid sample;

[0072] - Obtain a potential reading from the at least one ISE; and

[0073] - Determine the estimated ion activity based on the potential reading.

[0074] In the context of this invention, a blood sample is preferred as the body fluid sample. Blood samples are particularly advantageous because the preferred ion concentrations specified herein for the reference electrolyte closely approximate the typical ion concentrations of a blood sample. As used herein, the term "blood sample" refers to any sample that may be derived from the subject's blood, including but not limited to whole blood, serum, plasma, and any derivatives or portions thereof. Blood samples can be collected by any suitable method known in the art, including but not limited to venipuncture or finger-prick blood collection. Preferably, the blood sample is selected from the group consisting of whole blood, serum, and / or plasma.

[0075] In another embodiment, the body fluid sample is selected from urine, cerebrospinal fluid, and / or pleural fluid, especially urine. Furthermore, the body fluid sample can be any aqueous sample.

[0076] Preferably, the analyte is an ion, especially an ion with at least one ISE selectivity. For example, this method can be used to measure Cl in body fluid samples. - And the at least one ISE can be Cl - Selective electrode.

[0077] In a preferred embodiment, when the potential measurement system includes more than one ISE, the method includes the following steps:

[0078] - Contact each ISE in the ISE with the body fluid sample;

[0079] - Obtain potential readings from each ISE; and

[0080] - Determine the estimated ion activity based on the stated potential reading.

[0081] Specifically, the method may include the steps defined above for the processor of the potential measurement system.

[0082] To facilitate understanding of the present invention, several terms are defined below. The terms defined herein have the meanings commonly understood by one of ordinary skill in the art related to the present invention. Terms such as “a,” “an,” and “the” do not refer to a single entity but encompass general categories that can be illustrated with specific examples. Unless otherwise stated in the claims, the terms used herein are used to describe specific embodiments of the invention, but their use does not limit the invention.

[0083] Unless otherwise stated, all parameters used herein correspond to those under IUPAC SATP (“Standard Ambient Temperature and Pressure”) conditions, specifically a temperature of 25°C and a pressure of 101,300 Pa.

[0084] Unless otherwise specified as weight to weight (w / w) or otherwise, percentages (%) used herein refer to weight to volume (w / v).

[0085] This invention relates to the following preferred embodiments:

[0086] Example 1. A reference electrode for a potentiometric measurement system for measuring analytes in body fluid samples, the reference electrode comprising an Ag / AgCl conductor element in contact with a reference electrolyte, wherein chloride ions (Cl-) in the reference electrolyte... - The concentration of the reference electrolyte is between 50 mmol / L and 200 mmol / L, and the ionic strength of the reference electrolyte is between 75 mmol / L and 300 mmol / L.

[0087] Example 2. The reference electrode according to the foregoing examples, wherein the ionic strength of the reference electrolyte is between 80 mmol / L and 280 mmol / L, preferably between 85 mmol / L and 270 mmol / L, more preferably between 90 mmol / L and 260 mmol / L, more preferably between 95 mmol / L and 250 mmol / L, more preferably between 100 mmol / L and 240 mmol / L, more preferably between 105 mmol / L and 230 mmol / L, more preferably between 110 mmol / L and 220 mmol / L, more preferably between 115 mmol / L and 210 mmol / L, more preferably between 120 mmol / L and 200 mmol / L, more preferably between 125 mmol / L and 190 mmol / L, more preferably between 130 mmol / L and 180 mmol / L, more preferably between 135 mmol / L and 170 mmol / L, more preferably between 140 mmol / L. Between mmol / L and 160 mmol / L.

[0088] Example 3. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte contains chloride ions (Cl... - The concentration is between 55 mmol / L and 190 mmol / L, preferably between 60 mmol / L and 180 mmol / L, more preferably between 65 mmol / L and 170 mmol / L, more preferably between 70 mmol / L and 160 mmol / L, more preferably between 75 mmol / L and 150 mmol / L, more preferably between 80 mmol / L and 140 mmol / L, more preferably between 85 mmol / L and 130 mmol / L, more preferably between 90 mmol / L and 120 mmol / L, more preferably between 95 mmol / L and 110 mmol / L, and more preferably between 97 mmol / L and 105 mmol / L.

[0089] Example 4. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte contains sodium ions (Na... + The concentration is between 70 mmol / L and 270 mmol / L, more preferably between 80 mmol / L and 250 mmol / L, more preferably between 90 mmol / L and 230 mmol / L, more preferably between 100 mmol / L and 210 mmol / L, more preferably between 110 mmol / L and 190 mmol / L, more preferably between 120 mmol / L and 170 mmol / L, more preferably between 135 mmol / L and 155 mmol / L, and more preferably between 140 mmol / L and 148 mmol / L.

[0090] Example 5. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte contains potassium ions (K... + The concentration is between 2.1 mmol / L and 8.3 mmol / L, more preferably between 2.3 mmol / L and 7.9 mmol / L, more preferably between 2.5 mmol / L and 7.5 mmol / L, more preferably between 2.7 mmol / L and 7.1 mmol / L, more preferably between 2.9 mmol / L and 6.7 mmol / L, more preferably between 3.1 mmol / L and 6.3 mmol / L, more preferably between 3.3 mmol / L and 5.9 mmol / L, more preferably between 3.5 mmol / L and 5.5 mmol / L, and more preferably between 3.7 mmol / L and 5.1 mmol / L.

[0091] Example 6. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte contains calcium ions (Ca... 2+ The concentration is between 0.5 mmol / L and 2.5 mmol / L, more preferably between 0.6 mmol / L and 2.3 mmol / L, more preferably between 0.7 mmol / L and 2.1 mmol / L, more preferably between 0.8 mmol / L and 1.9 mmol / L, more preferably between 0.9 mmol / L and 1.7 mmol / L, more preferably between 1.0 mmol / L and 1.5 mmol / L, and more preferably between 1.08 mmol / L and 1.30 mmol / L.

[0092] Example 7. The reference electrode according to any of the foregoing embodiments, wherein the pH value of the reference electrolyte is between 6.5 and 8.5, more preferably between 6.6 and 8.3, more preferably between 6.7 and 8.1, more preferably between 6.8 and 8.0, more preferably between 6.9 and 7.9, more preferably between 7.0 and 7.8, more preferably between 7.1 and 7.7, more preferably between 7.2 and 7.6, more preferably between 7.3 and 7.5, and more preferably between 7.35 and 7.45.

[0093] Example 8. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte is at least partially immobilized.

[0094] Example 9. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte is in gel form.

[0095] Example 10. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte contains a gel forming agent, preferably a polysaccharide, more preferably agarose.

[0096] Example 11. A reference electrode according to any of the preceding embodiments, wherein the concentration of the gel forming agent in the reference electrolyte is between 0.2 wt.% and 20 wt.%, preferably between 0.5 wt.% and 10 wt.%, and more preferably between 1 wt.% and 4 wt.%.

[0097] Example 12. A reference electrode according to any of the preceding embodiments, wherein the reference electrolyte contains a humectant.

[0098] Example 13. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte comprises a polyol.

[0099] Example 14. The reference electrode according to the foregoing examples, wherein the polyol is selected from carbohydrates, sugar alcohols, glycerol and / or ethylene glycol; especially glycerol.

[0100] Example 15. A reference electrode according to any of the preceding embodiments, wherein the concentration of polyol in the reference electrolyte is between 30 wt.% and 80 wt.%, preferably between 40 wt.% and 70 wt.%.

[0101] Example 16. A reference electrode according to any of the foregoing embodiments, wherein the reference electrolyte contains a porous additive, preferably fumed silica.

[0102] Example 17. A reference electrode according to any of the preceding embodiments, wherein the reference electrolyte contains 10 wt.% to 90 wt.% water, preferably 15 wt.% to 80 wt.% water, more preferably 20 wt.% to 60 wt.% water, and even more preferably 25 wt.% to 40 wt.% water.

[0103] Example 18. A potential measurement system for measuring the potential of an analyte in a body fluid sample, the potential measurement system comprising a reference electrode as described in any of the preceding embodiments and at least one ion-selective electrode (ISE).

[0104] Example 19. A potential measurement system according to the foregoing embodiments, wherein the at least one ISE is selected from the group consisting of: chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+ Selective electrodes and / or potassium ions (K) + Selective electrode.

[0105] Example 20. A potential measurement system according to the foregoing embodiments, wherein the at least one ISE is a chloride ion (Cl... - Selective electrode.

[0106] Example 21. A potential measurement system according to the foregoing embodiments, wherein the at least one ISE is a sodium ion (Na+). + Selective electrode.

[0107] Example 22. A potential measurement system according to any of the foregoing embodiments, wherein the potential measurement system comprises at least two, preferably at least three, ISEs, wherein the ISEs are selected from the group consisting of chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+ Selective electrodes and / or potassium ions (K) +Selective electrode, preferably, wherein the potential measurement system includes at least chloride ion (Cl) electrodes. - Selective electrode and sodium ions (Na) + Selective electrode.

[0108] Example 23. A potential measurement system according to any of the foregoing embodiments, wherein the potential measurement system includes at least chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+ Selective electrode and potassium ion (K) + Selective electrode.

[0109] Example 24. A potential measurement system according to any of the foregoing embodiments, wherein the potential measurement system includes a processor configured to perform the following steps:

[0110] A. Obtain potential readings from at least one of the aforementioned ISEs;

[0111] B. Determine the estimated ion activity based on the potential reading;

[0112] C. Determine the estimated diffusion potential based on the estimated ion activity; and

[0113] D. Correct the estimated ion activity based on the estimated diffusion potential to obtain the corrected ion activity.

[0114] Example 25. A potential measurement system according to any of the foregoing embodiments, wherein the processor is configured to perform the following steps:

[0115] A. Obtain potential readings from each ISE in the ISE;

[0116] B. Determine the estimated ion activity based on each potential reading;

[0117] C. Determine the estimated diffusion potential based on the estimated ion activity; and

[0118] D. Correct the estimated ion activity based on the estimated diffusion potential to obtain the corrected ion activity.

[0119] Example 26. A potential measurement system according to any of the foregoing embodiments, wherein the processor is configured to further perform the following steps:

[0120] E. Determining the second-order estimated diffusion potential based on corrected ion activity; and

[0121] F. The corrected ion activity is further corrected based on the second-order estimated diffusion potential to obtain the second-order corrected ion activity.

[0122] Example 27. A potential measurement system according to any of the foregoing embodiments, wherein steps E. and F. are repeated during the iteration process.

[0123] Example 28. A method for potentiometric measurement of analytes in body fluid samples using the potentiometric measurement system described in any of the foregoing embodiments, the method comprising the following steps:

[0124] - Contact the at least one ISE with a body fluid sample;

[0125] - Obtain a potential reading from the at least one ISE; and

[0126] - Determine the estimated ion activity based on the potential reading.

[0127] Example 29. The method according to any of the foregoing embodiments, wherein the method includes the following steps:

[0128] - Contact each ISE in the ISE with the body fluid sample;

[0129] - Obtain potential readings from each ISE; and

[0130] - Determine the estimated ion activity based on the potential reading.

[0131] Example 30. The method according to any of the foregoing embodiments, wherein the method includes the steps defined in the foregoing embodiments regarding the processor of the potential measurement system.

[0132] Example 31. The method according to any of the preceding embodiments, wherein the body fluid sample is selected from blood samples, urine samples, cerebrospinal fluid samples and / or pleural fluid samples; especially blood samples.

[0133] Example 32. The method according to any of the preceding embodiments, wherein the body fluid sample is a blood sample, and wherein the blood sample is selected from the group consisting of whole blood, serum and / or plasma. Attached Figure Description

[0134] The present invention will be further illustrated by the following figures and examples, but is not limited thereto.

[0135] Figure 1 shows a schematic diagram of an embodiment of the potential measurement system. (A) is a cross-section of the system in a first plane. (B) is a cross-sectional view of the system in a second plane perpendicular to the first plane.

[0136] Figure 2 shows the results of a validation study using 694 patient samples, with (A) Na + and (B)Cl - It is measured as an analyte. Detailed Implementation

[0137] Figure 1 shows two vertical cross-sectional views of an exemplary embodiment of the potential measurement system of the present invention. The system includes a sensor housing 1 disposed on a sensor support base 2, forming a sample channel 3. A blood sample introduced along the flow direction 6 can contact four ISEs 4, 4', 4'', 4''' arranged along the sample channel 3. Potential readings from these ISEs can be obtained through conductive elements 5. The sample channel 3 is in contact with an Ag / AgCl conductor element 8 via a gel reference electrolyte 7. A reference signal from the Ag / AgCl conductor element 8 can be obtained through conductive elements 9. The electrolyte composition of the reference electrolyte 7 is similar to that of a typical blood sample, thereby minimizing concentration variations due to ion diffusion between the reference electrolyte 7 and the sample in the sample channel 3. A diffusion potential may be generated at the interface between the reference electrolyte 7 and the sample in the sample channel 3. Potential readings from the ISEs 4, 4', 4''' can be used to determine the estimated ion activity in the sample, which in turn can be used to estimate the diffusion potential at the interface. This estimated diffusion potential can be used to correct the potential reading from the ISE to obtain the corrected ion activity. These corrected ion activities can then be used again during iterations to estimate the diffusion potential more accurately.

[0138] Figure 2 shows the measurement of Na using 694 patient samples. + (Group A) and Cl - (Group B) Validation study results. Based on the estimated diffusion potential, the effects of different levels of mathematical correction were compared using the following compensation models: Compensation Model A (no correction), Compensation Model B (based only on Na). + Correction), C (based on Na) + and Cl - Correction), D (based on Cl) - Na + Ca 2+ and K + (Corrected) and E (based on Cl) - Na + Ca 2+ K + (and estimated residual anion correction). TAE lo = Total analytical error (p low = 0.025), TAE hi = Total analytical error (p high= 0.950), IQR (deviation) = interquartile range of deviation (the difference between the 75th percentile and the 25th percentile in the data).

[0139] Example 1. Validation study.

[0140] A validation study was conducted using a potential measurement system, which includes four ISEs (Cl... - Na + Ca 2+ and K + The reference electrode and the reference electrode are basically designed as shown in Figure 1. The reference electrode consists of an Ag / AgCl conductor element and a reference electrolyte.

[0141] The reference electrolyte consists of a mixture of glycerol and a buffer solution containing 145 mmol / L Na. + 105 mmol / L Cl - 4 mmol / L K + and 1.5 mmol / L Ca 2+ To facilitate immobilization, agarose and fumed silica were added.

[0142] The correction based on the estimated diffusion potential is performed as described above, specifically by using the main input of the ISE and an iterative process that converges to the final estimate to estimate the diffusion potential based on the measured ion activity and the interionic interactions associated with the Henderson equation.

[0143] To assess the impact of this mathematical correction, five different compensation models were compared:

[0144] - Model A: No correction is made based on the estimated diffusion potential; that is, the readings obtained directly from ISEs are used.

[0145] Model B: Based on a single cation (Na) + Correction is performed based on the estimated Na in the sample. + The activity is determined by the estimated diffusion potential, and this estimated diffusion potential is used to correct the potential reading for each ISE to obtain the corrected ion activity.

[0146] Model C: Based on a single cation (Na) + ) and single anion (Cl - Correction is performed based on Na in the sample. + and Cl - The estimated activity determines the estimated diffusion potential;

[0147] - Model D: Based on Cl - Na + Ca2+ and K + All four ions were corrected:

[0148] - Model E: Based on Cl - Na + Ca 2+ and K + All four ions and the residual anion concentrations determined as described above were corrected.

[0149] For the validation study, 694 blood samples from human subjects were measured using a potential measurement system. To determine measurement accuracy, all samples were also measured using the cobas® b221 system as a reference method.

[0150] Figure 2A and 2B The figures show Na + and Cl - The results obtained for the analyte. As can be seen from the figure, the interquartile range (IQR) decreases as the compensation model is extended from model A to model E. Similarly, the lower and upper limits of the total analytical error (TAE) become closer, indicating that the measurement results are more accurate.

[0151] Therefore, although satisfactory results were already achieved without any mathematical corrections based on the estimated diffusion potential, the results were significantly improved after such corrections were made. The more ions considered when estimating the diffusion potential, the greater the improvement.

Claims

1. A reference electrode for a potentiometric measurement system for measuring an analyte in a body fluid sample, the reference electrode comprising an Ag / AgCl conductor element (8) in contact with a reference electrolyte (7), wherein chloride ions (Cl) in the reference electrolyte (7) are present in the reference electrolyte (7). - The concentration of sodium ions (Na) in the reference electrolyte (7) is between 50 mmol / L and 200 mmol / L. + The concentration of the reference electrolyte (7) is between 70 mmol / L and 270 mmol / L, and the ionic strength of the reference electrolyte (7) is between 75 mmol / L and 300 mmol / L.

2. The reference electrode according to claim 1, wherein the reference electrolyte (7) contains potassium ions (K... + The concentration of calcium ions (Ca) in the reference electrolyte (7) is between 2.1 mmol / L and 8.3 mmol / L. 2+ The concentration is between 0.5 mmol / L and 2.5 mmol / L.

3. The reference electrode according to any one of claims 1 or 2, wherein the reference electrolyte (7) is at least partially immobilized.

4. The reference electrode according to any one of claims 1 to 3, wherein the reference electrolyte (7) comprises a gel forming agent, preferably a polysaccharide.

5. The reference electrode according to any one of claims 1 to 4, wherein the reference electrolyte (7) comprises a humectant, preferably a polyol.

6. The reference electrode according to any one of claims 1 to 5, wherein the reference electrolyte (7) comprises a porous additive, preferably fumed silica.

7. A potential measurement system for measuring the potential of an analyte in a body fluid sample, the potential measurement system comprising a reference electrode according to any one of the preceding claims and at least one ion-selective electrode (ISE) (4, 4', 4'', 4''').

8. The potential measurement system according to claim 7, wherein the at least one ISE (4, 4', 4'', 4''') is selected from the group consisting of chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+ Selective electrode and / or potassium ion (K) + Selective electrode.

9. The potential measurement system according to any one of claims 7 or 8, wherein the potential measurement system comprises at least chloride ions (Cl... - Selective electrode, sodium ions (Na) + Selective electrode, calcium ions (Ca) 2+ Selective electrode and potassium ion (K) + Selective electrode.

10. The potential measurement system according to any one of claims 7 to 9, wherein the potential measurement system includes a processor configured to perform the following steps: A. Obtain potential readings from at least one ISE (4, 4', 4'', 4'''); B. Determine the estimated ion activity based on the stated potential reading; C. Determine the estimated diffusion potential based on the estimated ion activity; and D. Correct the estimated ion activity based on the estimated diffusion potential to obtain the corrected ion activity.

11. The potential measurement system of claim 10, wherein the processor is configured to further perform the following steps: E. Determine the second-order estimated diffusion potential based on the corrected ion activity; and F. The corrected ion activity is further corrected based on the second-order estimated diffusion potential to obtain the second-order corrected ion activity.

12. The potential measurement system according to claim 11, wherein steps E. and F. are repeated during the iteration process.

13. A method for potentiometric measurement of an analyte in a body fluid sample using a potentiometric measurement system according to any one of claims 7 to 12, the method comprising the following steps: - Contact the at least one ISE (4, 4', 4'', 4''') with the body fluid sample; - Obtain potential readings from at least one ISE (4, 4', 4'', 4'''); and - Determine the estimated ion activity based on the stated potential reading.

14. The method of claim 13, wherein the method comprises the steps defined by the processor of any one of claims 10 to 12 with respect to the potential measurement system.

15. The method according to any one of claims 13 or 14, wherein the body fluid sample is a blood sample, preferably wherein the blood sample is selected from the group consisting of whole blood, serum and / or plasma.

Images