A glycated protein is a substance which is produced by the non-enzymatic and irreversible binding of the amino group of an amino acid constituting a protein, with the aldehyde group of a reducing sugar such as aldose. See e.g., U.S. Pat. No. 6,127,138. Such a non-enzymatic and irreversible binding reaction is also called “Amadori rearrangement,” and therefore the above-mentioned glycated protein may also be called “Amadori compound” in some cases.
Nonenzymatic glycation of proteins has been implicated in the development of certain diseases, e.g., diabetic complications and the aging process (Takahashi et al., J. Biol. Chem., 272(19):12505-7 (1997); and Baynes and Monnier, Prog. Clin. Biol. Res., 304:1-410 (1989)). This reaction leads to dysfunction of target molecules through formation of sugar adducts and cross-links. Considerable interest has focused on the Amadori product that is the most important “early” modification during nonenzymatic glycation in vitro and in vivo.
Various assays for glycated proteins are known. For example, U.S. Pat. No. 6,127,138 discloses that a sample containing a glycated protein is treated with Protease XIV or a protease from Aspergillus genus, thereafter (or while treating the sample with the above protease) FAOD (fructosyl amino acid oxidase) is caused to react with the sample so as to measure the amount of oxygen consumed by the FAOD reaction or the amount of the resultant reaction product, thereby to measure the glycated protein.
In another example, U.S. Pat. No. 6,008,006 discloses that the amount of glycated proteins in a sample can be quantified by reacting the sample with first a reagent which is a combination of a protease and a peroxidase and second with a ketoamine oxidase. U.S. Pat. No. 6,008,006 also discloses a kit which contains the combined peroxidase/protease enzyme reagent and also the ketoamine oxidase. U.S. Pub. No. 2005/0014935 discloses methods and kits for measuring amount of glycated protein using a chimeric amadoriase. U.S. Pub. No. 2003/0162242 and EP 1304385 A1 also disclose methods of selectively determining glycated hemoglobin.
Methods for determining percentage of glycated hemoglobin A1c that require a separate measurement of total hemoglobin in the samples have been described. When a chemistry analyzer is used to determine the value of percentage of glycated hemoglobin A1c, a dual channel format is required. In this format, two separate assays are performed to determine 1) glycated hemoglobin A1c concentration, and 2) total hemoglobin concentration in the samples; and followed by calculating the ratio of glycated HbA1c to total hemoglobin to obtain percentage of HbA1c. Methods of directly assaying percentage of glycated hemoglobin without a separate measurement of total hemoglobin are described in WO 2008/013874.
Various electrochemical biosensors are known to use specific enzyme reactions to determine the amount of an analyte that serves as the enzyme substrate through electrochemical detection methods. The most utilized biosensor is the biosensor for glucose test that utilizes glucose oxidase to react with glucose in the blood and to generate H2O2 for detection electrochemically using designed electrodes including working, counter and reference with or without a mediator or catalyst. Electrode sensors for H2O2 detection have been well developed and electrode strips for H2O2 detection have now become commercially available. See, e.g., Luo et al., Biosensors and Bioelectronics 22:482-488, 2006; Wang et al., Chem. Rev. 108:814-825, 2008; U.S. App. Pub. No. 2009/0090623.
Besides the glucose test, an HbA1c test has become another important test for better monitoring diabetic conditions. Glucose tests report the glucose levels in the blood for the particular moment, whereas HbA1c tests give an average glucose level of the past 3 months. By testing HbA1c levels, physicians can have a better understanding on how their patients' glucose levels were controlled in the past 3 months, which in turn helps doctors to make therapeutic decisions. Recently, there is a growing trend to use HbA1c test as a screen test for identification of diabetic conditions. Therefore, a HbA1c test device that is as simple as a glucose meter and can be used by patients as a self-test device is highly desirable.