CN110779914A - 基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法 - Google Patents
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Abstract
本发明公开了一种基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法。本发明的技术要点是，将四碳二元酸或五碳二元酸与血液样品混合，四碳二元酸或五碳二元酸与血红蛋白作用生成具有过氧化物酶性质的复合物；向前述体系中加入双氧水和四甲基联苯胺，溶液变蓝，根据血液中血红蛋白浓度不同，TMB颜色深浅也不一样，再根据TMB在波长652nm处吸光度值与血红蛋白浓度的关系，能计算得到待测血液样品中血红蛋白的含量；向体系中加入葡萄糖氧化酶和TMB溶液，根据血液中葡萄糖浓度不同，TMB显色程度不同，根据氧化态TMB在波长652nm处吸光度值与葡萄糖浓度的关系，能计算得到待测血液样品中葡萄糖的含量。
Description
技术领域
本发明属于化学生物传感以及生物检测技术领域，具体涉及一种基于血红蛋白-四碳、五碳二元酸复合物试剂盒的制备方法。
背景技术
血红蛋白是红细胞的主要组成部分，能与氧结合，运输氧和二氧化碳。血红蛋白含量能很好地反映贫血程度。血红蛋白增高、降低对临床检测某些疾病有着重大的参考价值，比如血红蛋白病理性增多，常见于严重的先天性及后天性心肺疾患和血管畸形，如法氏四联症、发绀型先天性心脏病、阻塞性肺气肿、肺源性心脏病、肺动脉瘘或肺静脉瘘及携氧能力低的异常血红蛋白病等；也见于某些肿瘤或肾脏疾病，如肾癌、肝细胞癌、肾胚胎瘤及肾盂积水、多囊肾等；病理性减少，常见于骨髓造血功能衰竭，如再生障碍性贫血、骨髓纤维化所伴发的贫血；因造血物质缺乏或利用障碍所致的贫血，如缺铁性贫血、叶酸及维生素B12缺乏所致的巨幼细胞性贫血；因红细胞膜、酶遗传性的缺陷或外来因素所致红细胞破坏过多而导致的贫血，如遗传性球形红细胞增多症、海洋性贫血、阵发性睡眠性血红蛋白尿、异常血红蛋白病、免疫性溶血性贫血、心脏体外循环的大手术或某些生物性和化学性等因素所致的溶血性贫血以及某些急性或慢性失血所致的贫血。所以，检测血液中血红蛋白的含量具有重要的科学意义。
目前，临床检测血红蛋白一般采用比色法测定，包括：氰化高铁血红蛋白(HiCN)测定法、十二烷基硫酸钠血红蛋白(SDS)测定法、叠氮高铁血红蛋白(HiN3)法、碱羟血红蛋白法、溴代十六烷基三甲胺(CTAB)血红蛋白测定法等。这些方法均需要新鲜采集的血液样品才可检测，且有些存在有毒公害问题，有些操作繁琐复杂，有些准确度低，因此需要一种试剂简单易得，无毒无公害，测定方法简便且准确度高的血红蛋白测定法。
发明内容
本发明的目的在于提供一种方法简便、绿色安全、成本低廉的基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法，可用于检测血红蛋白和血糖。
本发明的上述目的是通过如下的技术方案来实现的：本发明基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法，包括如下步骤：
(1)将四碳二元酸或五碳二元酸与不同的血液样品混合，四碳二元酸或五碳二元酸与血红蛋白作用生成具有过氧化物酶性质的复合物；
(2)向步骤(1)的体系中加入双氧水和四甲基联苯胺即TMB溶液，溶液变蓝，根据血液中血红蛋白浓度的不同，TMB的颜色深浅也不一样，再根据TMB在波长652nm处的吸光度值与血红蛋白浓度的关系，能计算得到待测血液样品中血红蛋白的含量；且检测血红蛋白时对待测血液样品没有时限性和温度的限制；
(3)向步骤(1)的体系中加入葡萄糖氧化酶和TMB溶液，根据血液中葡萄糖浓度的不同，血红蛋白与四碳或五碳二元酸复合物催化分解双氧水的含量不同，TMB显色程度不同，根据氧化态TMB在波长652nm处的吸光度值与葡萄糖浓度的关系，能计算得到待测血液样品中葡萄糖的含量。
具体的，步骤(1)中，四碳二元酸或五碳二元酸包括酒石酸、丁二酸、戊二酸和谷氨酸。
具体的，步骤(2)中，双氧水的浓度为2.5mmol/L，TMB的浓度为5mmol/L，两种溶液的体积比为1：1。
具体的，步骤(2)中，所述检测血红蛋白时对待测血液样品没有时限性和温度的限制是，测定血红蛋白的含量时，待测血液样品或者是新鲜的液体血样，或者是室内外常温下存放一年以内的干血迹，或者是高温下加热两小时的血迹。
本发明利用血红蛋白-四碳、五碳二元酸复合物催化分解双氧水的特性，再依据TMB显色程度与血红蛋白或双氧水浓度的关系，进而达到检测血红蛋白或葡萄糖目的；成功地制备出检测血红蛋白与葡萄糖的高灵敏度技术，且现象明显，操作简便，灵敏度高，绿色安全，成本低廉，又对血液的保存条件与时间无要求。
本发明检测血红蛋白为高灵敏度技术，它能取代试剂盒中酶联免疫吸附反应中的生物酶。四碳、五碳二元酸包括酒石酸、丁二酸、戊二酸和谷氨酸等，价格便宜，在价格、稳定性等方面有生物酶大分子无可比拟的优点，更重要的是，血红蛋白-四碳、五碳二元酸复合物不存在生物蛋白酶容易失去生物活性的问题，可长时间放置依然保持活性，显著提高检测的稳定性和时长性。另一方面，引入TMB不同状态下表现出不同颜色，建立可视化检测。
通过本发明的方法处理，能得到不同颜色的TMB溶液，这种颜色变化可根据双氧水的浓度进行调控，通过颜色或吸光度值的变化达到了对血红蛋白或葡萄糖的超痕量检测。与现有技术相比，本发明方法简单、成本低、灵敏度高、稳定性强，能够实现快速准确的检测；且血液放置较长时间也可进行检测，样品无论是新鲜血液或长时间放置的血液，还是干的血迹均能快速反应生成具有强过氧化物酶性质的复合物。基于此优势，不仅可以对新鲜血液中的血红蛋白进行快速检测，还可对时间久远的血案现场进行痕迹检查，快速定位案发现场血迹位置及其走向。既可定量检测血红蛋白或葡萄糖含量，也可定性检测血液痕迹，具有很广阔的应用前景。
附图说明
图1为血红蛋白不同状态下的圆二色谱图。
图2为验证血红蛋白-酒石酸具有过氧化物酶性质的显色图片；其中，a试管为酒石酸-血红蛋白显色，b试管为血红蛋白显色。
图3为血红蛋白与血红蛋白-酒石酸复合物催化双氧水氧化TMB生成的ox-TMB的在652nm处的紫外吸收谱图。
图4为验证不同外界条件对血红蛋白的影响图片；其中，从左向右的试管分别为血红蛋白在25℃、40℃、50℃、60℃、70℃、80℃、90℃、100℃水浴1h后与酒石酸复合物的显色。
图5为血红蛋白在25℃、40℃、50℃、60℃、70℃、80℃、90℃、100℃水浴1h后与酒石酸作用生成的复合物催化双氧水氧化TMB生成的ox-TMB的在652nm处的紫外吸收谱图。
图6是图5中Ⅰ处和放大图。
图7为血红蛋白-酒石酸复合物的类过氧化物酶性质的稳定性图。
图8为血红蛋白浓度与ox-TMB在652nm处吸光度的线性方程图。Ⅰ
图9为血糖浓度与ox-TMB在652nm处吸光度的线性方程图。
具体实施方式
下面是本发明的具体实施例。如无特别说明，所用药品和器具均为常规化学实验药品和器具。
实施例1：
血红蛋白-酒石酸复合物的制备。
向100μL 1mg/mL血红蛋白中加入100μL 0.03M酒石酸，混合均匀，即得血红蛋白-酒石酸络合物，其圆二色图如图1所示，加入酒石酸后，血红蛋白的二级结构发生了改变，结合图2、图3，加了酒石酸的血红蛋白的催化性显著增强，氧化铁TMB在652nm处的吸光度明显上升。将血红蛋白在不同温度下加热1h后再加酒石酸，复合物的催化性并未明显减弱，如图4、图5、图6所示，图5为血红蛋白与酒石酸复合物在不同温度下催化分解双氧水氧化TMB生成ox-TMB的紫外吸收谱图，因高温度对血红蛋白与酒石酸复合物的催化性几乎无影响，故90℃、100℃的ox-TMB的紫外吸收谱图会重合，图6为图5谱图中Ⅰ处圈中部分的放大图。将血液滴在A4纸上放置半年，再加酒石酸，依然可使双氧水，TMB体系变蓝，如图7中照片所示。以上实验说明血红蛋白的结构不影响其与酒石酸的络合，故而不影响血红蛋白-酒石酸的类过氧化物酶性质。
实施例2：
血红蛋白-戊二酸复合物用于对血液中血红蛋白的检测。
准备一块微孔板，设置标准孔和样品孔。往微孔板中各加50μL标准品(不同浓度血红蛋白与戊二酸络合物复合物)和50μL样品(稀释10倍血清)，50μL 2.5mM H2O2，50μL 2.5mMTMB，10分钟后测652nm处的吸光度。标准品最终浓度为0.01g/L、0.02g/L、0.04g/L、0.06g/L、0.08g/L、0.1g/L。以标准品的浓度为横坐标，吸光度为纵坐标做标准曲线，如图8所示。再将各样品组的吸光度代入标准曲线中得到血红蛋白浓度，结果如表1所示。
表1
| 样品编号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| Elisa参考值(g/L) | 0.21 | 0.341 | 0.255 | 0.385 | 0.3125 | 0.3605 | 0.341 | 0.585 | 0.334 |
| 测定值(g/L) | 0.19 | 0.301 | 0.198 | 0.334 | 0.286 | 0.321 | 0.267 | 0.62 | 0.401 |
实施例3：
血红蛋白-酒石酸复合物用于对血液中葡萄糖的检测。
准备12支离心管，编号为1-12，1号离心管为对照组。往2-12号离心管里分别加100μL浓度为11种不同浓度的葡萄糖溶液，再向其中加入100μL浓度为0.5mg/mL的葡萄糖氧化酶，并将其放入37℃水浴锅中，30分钟后分别向每个离心管中加800μL浓度为0.5mg/mL的血红蛋白-酒石酸，400μL浓度为5mM TMB，室温下放置35分钟，最后用紫外-可见分光光度计测ox-TMB的吸光度。葡萄糖的最终浓度为0.005、0.01、0.03、0.05、0.07、0.09、0.11、0.13、0.15、0.2、0.25mM。
以葡萄糖的最终浓度作为横坐标，ox-TMB的吸光度作为纵坐标，做标准曲线，如图9所示。再准备9支离心管，编号A-I，A号离心管作为对照组，往B-I号离心管里分别加100μL稀释十倍的血清，再向其中加入100μL浓度为0.5mg/mL的葡萄糖氧化酶，并将其放入37℃水浴锅中，30分钟后分别向每个离心管中加800μL浓度为0.5mg/mL的血红蛋白-酒石酸，400μL浓度为5mM TMB，室温下放置35分钟，最后用紫外-可见分光光度计或酶标仪测氧化铁TMB的吸光度。将各样品组的吸光度代入标准曲线中得到血清中的葡萄糖浓度，结果如表2所示。
表2
Claims (4)
1.一种基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法，其特征在于包括如下步骤：
(1)将四碳二元酸或五碳二元酸与不同的血液样品混合，四碳二元酸或五碳二元酸与血红蛋白作用生成具有过氧化物酶性质的复合物；
(2)向步骤(1)的体系中加入双氧水和四甲基联苯胺即TMB溶液，溶液变蓝，根据血液中血红蛋白浓度的不同，TMB的颜色深浅也不一样，再根据TMB在波长652nm处的吸光度值与血红蛋白浓度的关系，能计算得到待测血液样品中血红蛋白的含量；且检测血红蛋白时对待测血液样品没有时限性和温度的限制；
(3)向步骤(1)的体系中加入葡萄糖氧化酶和TMB溶液，根据血液中葡萄糖浓度的不同，血红蛋白与四碳或五碳二元酸复合物催化分解双氧水的含量不同，TMB显色程度不同，根据氧化态TMB在波长652nm处的吸光度值与葡萄糖浓度的关系，能计算得到待测血液样品中葡萄糖的含量。
2.根据权利要求1所述基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法，其特征在于：步骤(1)中，四碳二元酸或五碳二元酸包括酒石酸、丁二酸、戊二酸和谷氨酸。
3.根据权利要求1所述基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法，其特征在于：步骤(2)中，双氧水的浓度为2.5mmol/L，TMB的浓度为5mmol/L，两种溶液的体积比为1：1。
4.根据权利要求1所述基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法，其特征在于：步骤(2)中，所述检测血红蛋白时对待测血液样品没有时限性和温度的限制是，测定血红蛋白的含量时，待测血液样品或者是新鲜的液体血样，或者是室内外常温下存放一年以内的干血迹，或者是高温下加热两小时的血迹。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112763484A (zh) * | 2021-01-27 | 2021-05-07 | 南京医科大学 | 一种基于比色生物传感器检测谷胱甘肽和/或过氧化氢的方法 |
Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4077772A (en) * | 1977-05-31 | 1978-03-07 | Baxter Travenol Laboratories, Inc. | Method for the determination of hemoglobin in trace amounts |
| US4806468A (en) * | 1987-02-05 | 1989-02-21 | Becton, Dickinson And Company | Measurement of glycosylated hemoglobin by immunoassay |
| JPH02122267A (ja) * | 1988-11-01 | 1990-05-09 | Green Cross Corp:The | ヘモグロビン定量試薬キット及びそれを用いるヘモグロビン定量方法 |
| WO1997035030A1 (en) * | 1996-03-22 | 1997-09-25 | Stc Technologies, Inc. | A solution-based assay for peroxidatively-active substances in bodily fluids |
| US5688526A (en) * | 1993-03-18 | 1997-11-18 | Terumo Kabushiki Kaisha | Hemoglobin-encapsulating liposome and method for making the same |
| WO2002021142A1 (fr) * | 2000-09-07 | 2002-03-14 | Wako Pure Chemical Industries, Ltd. | Procede de quantification de l'hemoglobine totale et de la glycohemoglobine |
| JP2006081471A (ja) * | 2004-09-16 | 2006-03-30 | Toyobo Co Ltd | 生体成分測定用試薬及び測定方法 |
| CN1908669A (zh) * | 2006-08-22 | 2007-02-07 | 东南大学 | 血红蛋白芯片的制备及其电化学检测方法 |
| US20070154976A1 (en) * | 2003-11-19 | 2007-07-05 | Daiichi Pure Chemicals Co., Ltd. | Method of determining substrate contained in hemoglobin-containing sample |
| WO2008135139A1 (en) * | 2007-05-07 | 2008-11-13 | Universität Heidelberg | Quantification of hydrogen peroxide by triggered reconstitution of horseradish peroxidase using a modified hemin |
| US20100178660A1 (en) * | 2009-01-14 | 2010-07-15 | Abbott Laboratories | Methods and kits for detecting hemoglobin in test samples |
| US20130130391A1 (en) * | 2010-03-31 | 2013-05-23 | Hiroaki Taira | Method of analyzing hemoglobins |
| CN103492881A (zh) * | 2010-12-20 | 2014-01-01 | 反应实验室有限公司 | 测定方法 |
| CN104048957A (zh) * | 2014-05-14 | 2014-09-17 | 南昌大学 | 基于石墨烯量子点的类过氧化物酶催化特性的葡萄糖检测方法 |
| WO2016052612A1 (ja) * | 2014-10-01 | 2016-04-07 | 東洋紡株式会社 | 活性エステル化ポリ－γ－グルタミン酸を用いたヘモグロビンの測定方法 |
| EP3120155A1 (en) * | 2014-03-21 | 2017-01-25 | Maxim, Demetrios, Samuel | Portable instrument for in vitro detection and quantification of biomarkers |
| US20170328920A1 (en) * | 2014-11-18 | 2017-11-16 | Colgate-Palmolive Company | Detecting Blood |
| CN109239064A (zh) * | 2018-10-26 | 2019-01-18 | 湖南科技大学 | 一种含铜纳米棒复合物快速检测试剂盒的制备方法及应用 |
-
2019
- 2019-11-05 CN CN201911068995.4A patent/CN110779914B/zh active Active
Patent Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4077772A (en) * | 1977-05-31 | 1978-03-07 | Baxter Travenol Laboratories, Inc. | Method for the determination of hemoglobin in trace amounts |
| US4806468A (en) * | 1987-02-05 | 1989-02-21 | Becton, Dickinson And Company | Measurement of glycosylated hemoglobin by immunoassay |
| JPH02122267A (ja) * | 1988-11-01 | 1990-05-09 | Green Cross Corp:The | ヘモグロビン定量試薬キット及びそれを用いるヘモグロビン定量方法 |
| US5688526A (en) * | 1993-03-18 | 1997-11-18 | Terumo Kabushiki Kaisha | Hemoglobin-encapsulating liposome and method for making the same |
| WO1997035030A1 (en) * | 1996-03-22 | 1997-09-25 | Stc Technologies, Inc. | A solution-based assay for peroxidatively-active substances in bodily fluids |
| WO2002021142A1 (fr) * | 2000-09-07 | 2002-03-14 | Wako Pure Chemical Industries, Ltd. | Procede de quantification de l'hemoglobine totale et de la glycohemoglobine |
| US20070154976A1 (en) * | 2003-11-19 | 2007-07-05 | Daiichi Pure Chemicals Co., Ltd. | Method of determining substrate contained in hemoglobin-containing sample |
| JP2006081471A (ja) * | 2004-09-16 | 2006-03-30 | Toyobo Co Ltd | 生体成分測定用試薬及び測定方法 |
| CN1908669A (zh) * | 2006-08-22 | 2007-02-07 | 东南大学 | 血红蛋白芯片的制备及其电化学检测方法 |
| WO2008135139A1 (en) * | 2007-05-07 | 2008-11-13 | Universität Heidelberg | Quantification of hydrogen peroxide by triggered reconstitution of horseradish peroxidase using a modified hemin |
| US20100178660A1 (en) * | 2009-01-14 | 2010-07-15 | Abbott Laboratories | Methods and kits for detecting hemoglobin in test samples |
| US20130130391A1 (en) * | 2010-03-31 | 2013-05-23 | Hiroaki Taira | Method of analyzing hemoglobins |
| CN103492881A (zh) * | 2010-12-20 | 2014-01-01 | 反应实验室有限公司 | 测定方法 |
| EP3120155A1 (en) * | 2014-03-21 | 2017-01-25 | Maxim, Demetrios, Samuel | Portable instrument for in vitro detection and quantification of biomarkers |
| CN104048957A (zh) * | 2014-05-14 | 2014-09-17 | 南昌大学 | 基于石墨烯量子点的类过氧化物酶催化特性的葡萄糖检测方法 |
| WO2016052612A1 (ja) * | 2014-10-01 | 2016-04-07 | 東洋紡株式会社 | 活性エステル化ポリ－γ－グルタミン酸を用いたヘモグロビンの測定方法 |
| US20170328920A1 (en) * | 2014-11-18 | 2017-11-16 | Colgate-Palmolive Company | Detecting Blood |
| CN109239064A (zh) * | 2018-10-26 | 2019-01-18 | 湖南科技大学 | 一种含铜纳米棒复合物快速检测试剂盒的制备方法及应用 |
Non-Patent Citations (5)
| Title |
|---|
| ENIKANAGABABU JOSEPH: "Heme Degradation during Autoxidation of Oxyhemoglobin", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| XINYI HUA: "Rapid modification of hemoglobin heme to form enhanced peroxidase-like activity for colorimetric assay", 《BIOSENSORS AND BIOELECTRONICS:X》 * |
| 凌辉生: "修饰血红素提高血红蛋白类过氧化物酶活性", 《生物加工工程》 * |
| 席永清等: "血红蛋白模拟酶催化荧光法测定葡萄糖", 《分析化学》 * |
| 邓晓琴: "速率法测定血浆(尿液)游离血红蛋白", 《山西医学检验》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112763484A (zh) * | 2021-01-27 | 2021-05-07 | 南京医科大学 | 一种基于比色生物传感器检测谷胱甘肽和/或过氧化氢的方法 |
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