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(12) United States Patent (10) Patent No.: US 6,911,131 B2 Miyazaki Et Al

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(12) United States Patent (10) Patent No.: US 6,911,131 B2 Miyazaki Et Al USOO6911131B2 (12) United States Patent (10) Patent No.: US 6,911,131 B2 Miyazaki et al. (45) Date of Patent: Jun. 28, 2005 (54) BIOSENSOR 5,413,690 A 5/1995 Kost et al. ............... 205/777.5 5,747,669 A 5/1998 Suzuki ...................... 73/23.21 (75) Inventors: Shoji Miyazaki, Ehime (JP); Hiroyuki 6,059,946 A 5/2000 Yukawa et al. ........ 204/403.14 Tokunaga, Ehime (JP), Masaki FOREIGN PATENT DOCUMENTS Fujiwara, Ehime (JP); Takahiro Nakaminami, Kyoto (JP); Junko EP O608872 B1 8/1994 Nakayama, Ehime (JP); Eriko EP 0872728 A1 10/1998 Yamanishi, Ehime (JP) JP 2-102448 4/1990 JP 5-87767 4/1993 (73) Assignee: Matsushita Electric Industrial Co., JP 8-5O112 2/1996 Ltd., Osaka (JP) JP 9-89831 4/1997 es JP 1O-784O7 3/1998 (*) Notice: Subject to any disclaimer, the term of this JP 2000-657782000-96.79 1/200058 patent is extended or adjusted under 35 JP 2000-81.408 3/2000 U.S.C. 154(b) by 336 days. JP 2001-59846 6/2001 WO 95/03543 2/1995 (21) Appl. No.: 09/979,842 f f OTHER PUBLICATIONS (22) PCT Filed: Mar. 28, 2001 “Sugar Alcohol entry downloaded from http://en.wikipedi (86) PCT No.: PCT/JP01/02558 a.org/wiki/Sugar alcohol on Jan. 25, 2005.* S371 (c)(1) “Reducing Sugar” entry downloaded from http://en.wikipe (2), (4) fate. May 1, 2002 dia.org/wiki/Reducing Sugarl on Jan. 25, 2005.* s 9 "Sugar entry downloaded from http://en.wikipedia.org/ (87) PCT Pub. No.: WO01/73419 wiki/Sugarl on Jan. 25, 2005.* PCT Pub. Date: Oct. 4, 2001 (Continued) (65) Prior Publication Data Primary Examiner Alex Noguerola (74) Attorney, Agent, or Firm-Amster, Rothstein & US 2002/0179442 A1 Dec. 5, 2002 Ebenstein LLP (30) Foreign Application Priority Data (57) ABSTRACT Mar.Nov. 29, 9, 2000 (JP) ........................................................................................ 1234,53712-90362 In a bibiosensor forf measuring a specificifi substanceb in a liquiliquid Sample, one or a combination of Sugar alcohol, metallic Salt, (51) Int. Cl. ....................... G01N 27/327; G01N 21/00 organic acid or organic acid Salt which has at least one (52) U.S. Cl. ..................................... 204/403.14; 422/57 carboxyl group in a molecule, and organic acid or organic (58) Field of Search ....................... 204/403.01-403.14; acid Salt which has at least one carboxyl group and one 422/57, 68.1 amino group in a molecule, is included in a reagent layer provided on electrodes, thereby providing a highly-accurate (56) References Cited biosensor which is excellent in Stability and has high response (Sensitivity, linearity) of the Sensor to the Substrate U.S. PATENT DOCUMENTS concentration. 5,242,793 A * 9/1993 Kariyone et al. ...... 204/403.11 5,397.451 A 3/1995 Senda et al. ............. 204/403.1 12 Claims, 7 Drawing Sheets 6 Y -O-conventional sensor 5,5- - A -invention sensor (actitol:25mM) s --invention sensor (naltitol:25mM) g 5 s > 3 4.5 C Cl ge h- 4. d 3.5 3 i -- i O s O 15 20 25 3O retention period (Hr) US 6,911,131 B2 Page 2 OTHER PUBLICATIONS Sorbital entry obtained from ACS Registry on STN.* Trehalose entry obtained from ACS Registry on STN.* Lactitol entry obtained from ACS Registry on STN.* Sucrose entry obtained from ACS Registry on STN.* Maltitol entry obtained from ACS Registry on STN.* Glycerol entry obtained from ACS Registry on STN.* JPO computer translation of JP 2000–081408 A (Yugawa et Mannitol entry obtained from ACS Registry on STN.* al.)1).* Ribose entry obtained from ACS Registry on STN.* * cited by examiner U.S. Patent Jun. 28, 2005 Sheet 1 of 7 US 6,911,131 B2 Fig.1 1 8 -O-Conventional sensor 1 6 -A-invention sensor (lactitol:5mM) 2 --invention sensor ((lactitol:10mM) 1 4. -x-invention sensor (lactitol:25mM) O 1 2 --invention sensor (lactitol:50mM) 1-1 O a 211. O 1OO 200 300 400 500 600 glucose concentration (mg/dL) --invention sensor (maltitol:10mM) aC -Yé-invention sensor (maltitol:25mM) --invention sensor (mattitol:50mM) O 100 200 300 400 500 600 700 glucose concentration (mg/dL) U.S. Patent Jun. 28, 2005 Sheet 2 of 7 US 6,911,131 B2 Fig.3 -O-Conventional sensor -A-invention sensor (lactitol:25mM) C -Hinvention sensor eu O O 5 1O 5 20 25 3O retention period (Hr) Fig.4 -O-conventional sensor retention period (Hr) U.S. Patent Jun. 28, 2005 Sheet 3 of 7 US 6,911,131 B2 Fi 9. 5 1.2 Oconventional sensor 0.0. 68 O - O O 5 10 15 20 25 30 retention period (Hr) Fig.6 -O-conventional Sensor - A invention sensor (magnesium sulfate:25mM) o 5 10 15 20 25 30 retention period (Hr) U.S. Patent Jun. 28, 2005 Sheet 4 of 7 US 6,911,131 B2 Fig.7 O 5 10 15 20 25 30 35 retention period (day) -O-Conventional SensOr -A-invention sensor (malonic acid) --invention sensor (glutaric acid) -X-invention sensor (adipic acid) Fig.8 O 5 1 O 15 20 25 30 35 retention period (day) -O-Conventional Sensor -A-invention sensor (benzoic acid) -O-invention sensor (phthalic acid) ->{-invention sensor (malic acid) U.S. Patent Jun. 28, 2005 Sheet 5 of 7 US 6,911,131 B2 Fig.9 16 14 1 2 1 O 8 6 4 2 -O-Couventional sensor 1. - A -invention sensor (Gly) 2 --invention sensor (Ser) -be-invention sensor (Pro) O O 1 OO 200 300 400 500 6OO 700 800 glucose Concentration (mg/dl) Fig.10 16 14 11 O2 8 6 -O-Couventional Sensor 4. -A-invention sensor (Thr) 2 --invention sensor (Lys-HCl) 2 H4 -(-invention sensor (sarcosine) --X--invention sensor (taurine) O OO 2OO 300 400 500 600 700 800 glucose Concentration (mg/dl) U.S. Patent Jun. 28, 2005 Sheet 6 of 7 US 6,911,131 B2 Fig.11 | : 5 I : (YNSSSS S& 7 % 12 N-2 37 11 U.S. Patent Jun. 28, 2005 Sheet 7 of 7 US 6,911,131 B2 Fig.12 US 6,911,131 B2 1 2 BIOSENSOR temperature and humidity where the temperature is over 30 C. and the humidity is over 80%, a reduction reaction occurs This application is the national stage under 35 U.S.C. 371 between the electron transfer agent and a portion of enzyme of International Application No. PCT/JP01/02558, filed on protein or hydrophilic polymer which is included in the Mar. 28, 2001. reagent layer 5 or 105, thereby generating a background electric current (noise electric current). As the value of the TECHNICAL FIELD background electric current increases with time, the Sensor performance is deteriorated. The present invention relates to a biosensor for analyzing a specific component in a liquid Sample and, more Furthermore, as a means to Solve the problem, it is possible to eliminate moisture and prevent the deterioration particularly, to a reagent formulation for composing a of the Sensor performance by enclosing a desiccant Such as reagent layer of the biosensor. Silica gel or activated alumina into a biosensor preservation BACKGROUND ART container which employs a molded container of resin or aluminium Seal. However, it is impossible to completely A biosensor is a Sensor which utilizes the molecule 15 eliminate water of molecular level remaining in the reagent identifying abilities of organic materials Such as included in the biosensor, with the desiccant alone. microorganisms, enzymes, and antibodies, and applies the Further, it is extremely hard to keep the preservation organic materials as molecular recognition elements. That is, container free of moisture penetration over long term, and the biosensor utilizes a reaction which occurs when an the reduction reaction between a portion of enzyme protein immobilized organic material recognizes a target Specific or hydrophilic polymer and the electron transfer agent is Substance, Such as oxygen consumption by respiration of a promoted when only a slight amount of moisture exists. microorganism, an enzyme reaction, and luminescence. Therefore, it is extremely difficult to effectively suppress the Among biosensors, enzyme Sensors have been put to increase in the background electric current with time. practical use. For example, enzyme Sensors for glucose, Further, when an inorganic Salt Such as potassium ferri lactic acid, cholesterol, lactose, urea, and amino acid are 25 cyanide is included in the mixed reagent layer composed of utilized in medical measurement or food industry. An various reagents Such as an enzyme and an electron transfer enzyme Sensor reduces an electron acceptor by an electron agent, the reagent layer is extremely easily crystallized in the generated by a reaction between a Substrate included in a process of drying the reagent Solution, whereby the Surface Sample Solution as a specimen and an enzyme, and a of the reagent layer becomes rough and uneven, resulting in measuring device electrochemically measures the reduction deterioration in the response (linearity, Sensitivity) of the quantity of the electron acceptor, thereby performing quan Sensor to the Substrate concentration and the measurement titative analysis of the Specimen. As an example of Such accuracy. biosensor, a Sensor proposed in Japanese Published Patent The present invention is made to solve the above Application No. Hei. 11-1324,511 has been known. mentioned problems and has for its object to provide a FIGS. 11 and 12 are exploded perspective views illustrat 35 highly-accurate biosensor which efficiently prevents dete ing conventional biosensors for measuring a blood Sugar rioration of the performance of the biosensor due to contact level. A measuring electrode 2 or 102 (also referred to as a with moisture, and has high response (linearity, Sensitivity) working electrode), a counter electrode 3 or 103, and a of the Sensor to the Substrate concentration.
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