CN112098464A - 微阵列糖类代谢分析检测装置及方法 - Google Patents
微阵列糖类代谢分析检测装置及方法 Download PDFInfo
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Abstract
本发明提供一种微阵列糖类代谢分析检测装置,该装置包括:控制单元、信号采集单元、微阵列传感器单元和微量注射泵,控制单元连接微量注射泵和信号采集单元,微阵列传感器单元连接信号采集单元;微量注射泵受控制单元控制向微阵列传感器单元定量加入糖溶液,信号采集单元采集微阵列传感器单元受糖溶液刺激产生的电信号,信号采集单元向控制单元输送数据,控制单元记录和处理数据。本发明的微阵列糖类代谢分析检测装置灵敏度高、稳定性好,制备与操作简单。
Description
技术领域
本申请涉及生物传感器领域,尤其涉及微流控糖类代谢分析检测装置及方法。
背景技术
味觉是食物在人的口腔内对味觉器官化学感受系统进行刺激而产生的一种感觉,包括甜、酸、苦、咸四种基本味觉。人们平常尝到的各种味道,都是这四种味觉混合的结果。味觉的感受器是舌头上的味蕾,味蕾呈卵圆形,主要由味觉细胞和支持细胞组成,味觉细胞顶部有微绒毛向味孔方向伸展,与唾液接触,细胞基部有神经纤维支配。食物中的化学物质溶解在唾液中,通过味孔与味觉感受器细胞接触,在那里它们与味觉感受器或离子通道相互作用。这些相互作用触发细胞内的信号级联,诱导细胞的动作电位,电信号最终通过神经纤维传递到大脑,形成味觉。味觉细胞表面有许多味觉感受分子,不同物质能与不同的味觉感受分子结合而呈现不同的味道。人的味觉从受食物刺激到感受到滋味仅需1.5-4.0ms,比视觉13-45ms,听觉1.27-21.5ms,触觉2.4-8.9ms都快。一直以来,味觉的分析主要采用人工味觉分析法来判断,具有较大的主观性,难以精准分析评价食品带来的味觉。因此,通过味觉传感器能够实现现场、快速、实时检测,对于食品领域具有重要的意义。尤其需要在进食导致的流动状态下实现动态检测更为必要。
糖是一种重要的调味品,根据所含热量分为高热量甜味剂和低热甜味剂。高热量甜味剂包括蔗糖、蜂蜜等天然甜味剂,所含热量高,长期超量食用容易引起肥胖甚至糖尿病。低热甜味剂是指具有甜味、产生热能低且其营养价值低的物质,常用于控制血糖升高,避免肥胖,控制体重和预防心血管疾病的发生,还用作糖尿病患者食糖代用品。人类和其他哺乳动物的口腔中乳酸菌与食物中的微量糖发酵反应产生有机酸,包括乳酸、乙酸(乙醇)等产物,赋予不同糖类特定的口味。通过生物传感器准确分析乳酸菌对不同糖类的代谢特性,对于食品领域制备低热量且口味趋近蔗糖的甜味剂具有重大意义。
发明内容
本发明的目的是针对现有技术存在的缺点,提出一种微阵列糖类代谢分析检测装置及方法,解决现有技术中存在的常规味觉分析检测主观性强、检测物静止带来的准确性差等问题。
本发明提供一种微阵列糖类代谢分析检测装置,该装置包括:控制单元、信号采集单元、微阵列传感器单元和微量注射泵,控制单元连接微量注射泵和信号采集单元,微阵列传感器单元连接信号采集单元;微量注射泵受控制单元控制向微阵列传感器单元定量加入糖溶液,信号采集单元采集微阵列传感器单元受糖溶液刺激产生的电信号,信号采集单元向控制单元输送数据,控制单元记录和处理数据。
优选的,控制单元包括温度控制模块,用于控制检测温度。
优选的,信号采集单元包括多通道放大器和模数转换电路,多通道放大器用于放大微阵列传感器不同电极的检测信号,模数转换电路用于将检测信号转换为模拟信号。
优选的,微阵列传感器单元包括微电极阵列、检测腔和味觉活性层;
优选的,检测腔固定在微电极阵列上,味觉活性层位于检测腔内,与微电极阵列上表面贴合。微电极阵列包括正多边形节点等间距设置的电极阵列。微电极阵列的电极间距与味觉活性单元密度相适应。
优选的,味觉活性层包括具有生物活性的味觉细胞。味觉活性层以味觉细胞的味孔朝上的方式与微电极阵列上表面贴合。在味觉活性层上设置张紧拉伸的PMMA网,使味觉活性层与微电极阵列紧密贴合。
优选的,在味觉活性层和微电极阵列间形成有多聚鸟氨酸和层粘素层。
本发明还提供一种利用上述装置进行糖类代谢分析检测的方法,包括:用缓冲液冲洗测试腔内的味觉活性层,用显微镜观察确定被味觉活性层中的味觉细胞覆盖的电极并对微阵列电极的微电极进行编号;将糖溶液泵入检测腔;实时记录微阵列电极的多路通道测试信号,并根据检测信号描绘该糖溶液的乳酸菌代谢曲线。
优选的,缓冲液中包含1.0×107CFU/ml的乳酸菌;糖溶液的泵送速度为300-600mL/h,检测温度为37℃-38℃。
优选的,没有味觉细胞耦合的电极输出的信号作为测试基线,有味觉细胞耦合电极输出的测试信号。
优选的,不同糖溶液注入的最小时间间隔为5分钟;在两种糖溶液检测之间,用新鲜标准灌流液冲洗测试腔,再用缓冲液冲洗测试腔。
优选的,糖溶液包括75mmol/L蔗糖溶液,以及含有三氯蔗糖、赤藓糖醇、麦芽糖醇和维生素C的、甜度与75mmol/L蔗糖溶液一致的且酸度为pH3.0-4.0的混合溶液两种糖溶液。
与现有技术相比,本发明实施例的有益效果是:本发明通过微电极阵列与味觉活性层有机结合获得高灵敏度、高稳定性的微阵列传感器,简化味觉传感器制备与操作;通过PMMA网、多聚鸟氨酸和层粘素层固定味觉细胞,实现味觉活性层有效固定,提高传感器检测的稳定性;基于本发明的糖类乳酸装置,进行糖类口味模拟检测,准确获得代谢口味与蔗糖接近的复合甜味剂的酸度范围。
附图说明
通过下面结合附图对其示例性实施例进行的描述,本发明上述特征和优点将会变得更加清楚和容易理解。
图1是本发明的微阵列糖类代谢分析检测装置的结构示意图。
图2是本发明的微阵列传感器单元的结构示意图。
图3是本发明的微阵列糖类乳代谢分析检测方法的流程图。
具体实施方式
下面结合附图对本发明作进一步详细说明,以便于本领域技术人员准确理解本发明。
实施例1
如图1所示,本实施例提供的微阵列糖类代谢分析检测装置100,包括:控制单元110、信号采集单元120、微阵列传感器单元130和微量注射泵140。微量注射泵140与控制单元110连接,受控制单元110控制往微阵列传感器单元130定量加入检测用糖溶液,微阵列传感器单元130与信号采集单元120连接,实现微阵列传感器单元130的信号采集。信号采集单元120通过串口与控制单元110相连,完成数据传输、记录和处理。
微量注射泵140可通过异步接收/发送协议(UART)控制检测溶液的注射量。信号采集单元120包括多通道放大器,用于放大微阵列传感器单元130各通道电压信号,信号采集单元120还包括模数转换点路,用于将检测到的电压信号转换位数字信号,传输给控制单元110。微阵列糖类代谢分析检测装置100还控制单元110包括温度控制模块,用于控制分析检测的温度,通常在37℃-38℃。
[微阵列传感器单元]
如图2所示,本实施例微阵列传感器单元130包括微电极阵列131、检测腔132和味觉活性层133。微电极阵列131包括正多边形节点等间距电极阵列,从而避免过多电极导致干扰增大、引线设置困难和减少并行分析的复杂性。电极间距与味觉活性单元密度相适应,以减少相邻味觉活性单元的干扰。检测腔132固定在微电极阵列131上,味觉活性层133位于检测腔132内,与微电极阵列131上表面贴合。微阵列传感器单元130的制备工艺流程包括:
步骤S110:选用450μm厚的硅基板,使用丙酮清洗,用200℃加热30min烘干后,用激光切割成扁长为6mm的硅片;
步骤S120:在硅片上涂布一层厚度为20μm-150μm的光刻胶膜(例如负性光刻胶),用阶梯法烘干,在65℃保持1分钟,升温至95℃下驳斥5-10分钟,降温至65℃保持1分钟,然后静置冷却;
步骤S130:采用波长为365nm,光强为13mw/cm2的光进行曝光,时间为5-30秒,掩模上的图案为6×6分布的圆孔阵列,圆孔直径为5μm-50μm,圆孔间距为直径的1-2倍,圆孔为通孔;
步骤S140:在65℃保持1分钟,升温至95℃下驳斥5-10分钟,然后静置冷却,用显影剂显影5秒-2分钟,显影剂为PGMEA,在150℃固化5-10分钟;
步骤S150:用真空蒸镀、真空溅射、真空离子镀或化学气相沉积法在带有圆孔阵列的硅片上填充圆孔并形成20nm-80nm厚度的导电层,导电层选用金、银、铜、镍、钛、铂等任一种;
步骤S160:用干式蚀刻形成电极阵列及各电极的引导线,形成电极阵列芯片;
步骤S170:用具有生物稳定性的胶直径将参测腔固定在电极阵列芯片的表面上,检测腔为边长5mm的PMMA方框;
步骤S180:将带有味觉活性组织层放入检测腔,以味孔朝上的方式与电极阵列芯片上表面贴合,味觉神经与电极连通,通过味觉活性组织层上设置张紧拉伸的PMMA网使组织层与电极阵列紧密贴合,促进电性连接。
为了促进味觉活性层133与微电极阵列131贴合,可在室温下将器件浸入含有0.1mg/ml的多聚鸟氨酸的磷酸盐缓冲液中24小时,再浸入含8g/ml层粘素的磷酸盐缓冲液中24小时,在味觉活性层和微电极阵列间形成4nm的多聚鸟氨酸和层粘素层,因多聚鸟氨酸和层粘素带正电荷,可促进味觉活性层粘附在微电极阵列上。
[味觉活性层]
本实施例的味觉活性层,通过分离老鼠的舌上皮组织,获得具有完整味觉细胞,选用体重约250g的SD大鼠。具体制备流程如下:
步骤S210:固定SD大鼠,在腹部注射乌拉坦麻醉;
步骤S220:切除舌头到环状乳头近端,剥离舌菌状乳头,用林格氏液孵化并冲洗三次;
在1000ml蒸馏水中加入8.6g氯化钠,0.3g氯化钾,0.28g氯化钙制备林格氏液。
步骤S230:注0.4ml射缓冲液到舌上皮下;
缓冲液含有1.5mg/mlII型胶原酶(Gibco)和3mg/mlII型消旋酶(Roche)。
步骤S240:在室温下用林格氏液孵化8-10分钟后,将舌上皮从下层肌层剥离,放入林格氏液中;
步骤S250:将舌上皮分离为5mm×5mm的规格,并用林格氏液漂洗,得到味觉活性层。
实施例2
乳酸菌对糖的分解代谢主要包括两种途径,即糖酵解途径(同型乳酸发酵)和磷酸解酮酶途径(异型乳酸发酵)。糖酵解途径(embden-meverhef-parnus pathway,EMP)是指葡萄糖在无氧条件下酵解生成乳酸和三磷酸腺苷(ATP)。磷酸解酮酶途径(phosphoketolasepathway,PK)是指葡萄糖在无氧条件下酵解生成等量的乳酸、乙醇、CO2和ATP。因此,乳酸菌对糖代谢都会产生对口味有影响的乳酸,采用本发明提供的糖类乳酸菌代谢分析检测装置,通过检测pH的变化来监测乳酸菌对不同糖的分解代谢情况。如图3所示,本实施例提供一种利用实施例1的微阵列糖类代谢分析检测装置对糖类代谢进行分析检测的方法,具体步骤如下:
步骤S310:用缓冲液冲洗测试腔里的味觉活性层,对微阵列电极的微电极进行编号,用显微镜观察确定有味觉细胞覆盖的电极;
缓冲液中包含1.0×107CFU/ml的乳酸菌。
步骤S320:将糖溶液泵入检测腔;
糖类可以是天然甜味剂,也可以是人工甜味剂,可以是单一种类甜味剂,也可以是复合种类甜味剂,也可以是添加其他调味剂的功能甜味剂。人工甜味剂包括高甜度甜味剂和甜味缓冲剂。其他调味剂可以为包括有机酸或无机酸在内的酸味添加剂。
高甜度甜味剂是指具有高于蔗糖、果糖或葡萄糖的增甜效能,而且热量较低的物质。非限制性实例包括:环己基氨基磺酸钠(甜蜜素)、环己基氨基磺酸钙、L-α-天冬氨酰-N-(2,2,4,4-四甲基-3-硫化三亚甲基)-D-丙氨酰胺(阿力甜)、天门冬酰苯丙氨酸甲酯乙酰磺胺酸、三氯蔗糖(蔗糖素)、乙酰磺胺酸钾(安赛蜜)、天门冬酰苯丙氨酸甲酯(阿斯巴甜)、糖精、新橘皮苷二氢查酮、N-[N-(3,3-二甲基丁基)]-L-α-天门冬氨-L-苯丙氨酸1-甲酯(纽甜)、莱包迪苷A、莱包迪苷B、莱包迪苷C、莱包迪苷D、莱包迪苷E、杜尔可苷A、甜菊(糖)苷、罗汉果苷Ⅳ(罗汉果皂苷)、罗汉果苷Ⅴ(罗汉果皂苷)、罗汉果甜味剂(罗汉果甜苷)、赛门苷、甘草酸三钾盐、甘草酸三钠盐、甘草酸单铵盐、甘草酸铵、甘草酸一钾、甘草酸三钾、奇异果甜蛋白(索马甜)、仙茅甜蛋白、应乐果甜蛋白(莫奈林)、马槟榔甜蛋白、博灵(巴西甜蛋白)、荷南度辛、甘茶素、奥斯来丁、聚婆朵苷、培它丁、环拉酸盐、甘草皂苷、乙酰舒泛钾、阿司帕坦等。
甜味缓冲剂包括具有低于蔗糖、果糖或葡萄糖或与其相当的增甜效能,而且热量较低的物质,例如糖类还原形式的糖醇、多醇或聚醇,其中该羰基(醛或酮,还原性糖类)已被还原成初级或次级羟基。本发明甜味缓冲剂的非限制性实例包括:木糖醇、山梨糖醇、D-甘露糖醇、麦芽糖醇、异麦芽糖醇、赤藓糖醇、半乳糖醇、乳糖醇(4-β-D吡喃半乳糖-D-山梨醇);棉籽糖、乳糖、麦芽糖、异麦芽酮糖、α-D-葡萄糖、α-D-甘露糖、α-D-木糖、α-D-半乳糖、β-D-呋喃果糖、β-D-麦芽糖、β-D-乳糖;明胶,酪蛋白酸钠,阿拉伯胶、罗望子胶、田菁胶、琼脂、海藻酸钠、海藻酸钾,卡拉胶、果胶、黄原胶、β-环糊精、羧甲基纤维素钠、淀粉磷酸酯钠、羧甲基淀粉钠、羟丙基淀粉或海藻酸丙二醇酯。
有机酸添加剂包括任何含有-COOH基团之化合物,包括但不限于C2-C30羧酸类、取代的羟基C1-C30羧酸类、取代的肉桂酸类、羟基酸类、取代的羟基苯甲酸类、取代的环己基羧酸类、鞣酸、乳酸、酒石酸、柠檬酸、延胡索酸、葡萄糖酸、羟基柠檬酸、苹果酸、富马酸、马来酸、琥珀酸、水杨酸、肌酸、醋酸、抗坏血酸、己二酸、乙酸、乙二酸、正丁酸、甲酸、聚谷氨酸、盐酸葡萄糖胺、葡萄糖酸-δ-内酯及其碱金属或碱土金属盐类衍生物。
无机酸添加剂包括但不限于磷酸、亚磷酸、聚磷酸、碳酸、磷酸二氢钠、及其对应之碱金属或碱土金属盐类(如六磷酸肌醇镁/钙)。
糖溶液的输送速度为300-600mL/h。
步骤S330:实时记录微电极阵列的多路通道测试信号,并根据检测信号描绘该糖溶液的乳酸菌代谢曲线。
工作温度为37℃-38℃,连续记录电压信号5分钟,没有味觉细胞耦合的电极输出的信号为环境噪声和系统噪声,作为测试基线,有味觉细胞耦合电极输出的信号作为测试信号。
在两种糖溶液检测之间需要用新鲜标准灌流液将糖溶液刺激物从测试腔内冲洗掉,最后用包含1.0×107CFU/ml乳酸菌的缓冲液冲洗。为了让电极和味觉组织恢复到稳定状态,减少残余味觉物质对测试的影响,不同糖溶液注入的最小时间间隔为5分钟。
[糖类代谢口味比较实例]
采集时间为5分钟,温度为36℃。测试的糖类溶液包括:
标准液:75mmol/L蔗糖溶液。
酸度梯度测试液各成分的具体含量如表1所示。
表1:测试液成分含量
使用数字防水pH计,HM Digital pH计PH-200,测量样品pH值。pH计用GeneralHydroponics生产的标准pH7.0参比溶液进行校准。该设备在测量前校准。
结果显示随着时间的推移,蔗酸度范围在3.0-4.0之间的测试液,在120-180秒间与蔗糖代谢吻合度更高。
以上通过实施例对于本发明的发明意图和实施方式进行详细说明,但是本发明所属领域的一般技术人员可以理解,本发明以上实施例仅为本发明的优选实施例之一,为篇幅限制,这里不能逐一列举所有实施方式,任何可以体现本发明权利要求技术方案的实施,都在本发明的保护范围内。
需要注意的是,以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施方式仅限于此,在上述实施例的指导下,本领域技术人员可以在上述实施例的基础上进行各种改进和变形,而这些改进或者变形落在本发明的保护范围内。
Claims (15)
1.一种微阵列糖类代谢分析检测装置,其特征在于,该装置包括:控制单元、信号采集单元、微阵列传感器单元和微量注射泵,所述控制单元连接所述微量注射泵和所述信号采集单元,所述微阵列传感器单元连接所述信号采集单元;
所述微量注射泵受所述控制单元控制向所述微阵列传感器单元定量加入糖溶液,所述信号采集单元采集所述微阵列传感器单元受糖溶液刺激产生的电信号,所述信号采集单元向所述控制单元输送数据,所述控制单元记录和处理所述数据。
2.根据权利要求1所述的微阵列糖类代谢分析检测装置,其特征在于:所述控制单元包括温度控制模块,用于控制检测温度。
3.根据权利要求1所述的微阵列糖类代谢分析检测装置,其特征在于:所述信号采集单元包括多通道放大器和模数转换电路,所述多通道放大器用于放大所述微阵列传感器不同电极的检测信号,所述模数转换电路用于将所述检测信号转换为模拟信号。
4.根据权利要求1所述的微阵列糖类代谢分析检测装置,其特征在于:所述微阵列传感器单元包括微电极阵列、检测腔和味觉活性层;
所述检测腔固定在所述微电极阵列上,所述味觉活性层位于所述检测腔内,与所述微电极阵列上表面贴合。
5.根据权利要求4所述的微阵列糖类代谢分析检测装置,其特征在于:所述微电极阵列包括正多边形节点等间距设置的电极阵列。
6.根据权利要求5所述的微阵列糖类代谢分析检测装置,其特征在于:所述微电极阵列的电极间距与所述味觉活性单元密度相适应。
7.根据权利要求4所述的微阵列糖类代谢分析检测装置,其特征在于:所述味觉活性层包括具有生物活性的味觉细胞。
8.根据权利要求7所述的微阵列糖类代谢分析检测装置,其特征在于:所述味觉活性层以味觉细胞的味孔朝上的方式与所述微电极阵列上表面贴合。
9.根据权利要求8所述的微阵列糖类代谢分析检测装置,其特征在于:在所述味觉活性层上设置张紧拉伸的PMMA网,使所述味觉活性层与所述微电极阵列紧密贴合。
10.根据权利要求8所述的微阵列糖类代谢分析检测装置,其特征在于:在所述味觉活性层和所述微电极阵列间形成有多聚鸟氨酸和层粘素层。
11.一种利用权利要求1-10任意一项所述的装置进行糖类代谢分析检测的方法,其特征在于,该方法包括:
用缓冲液冲洗测试腔内的味觉活性层,用显微镜观察确定被味觉活性层中的味觉细胞覆盖的电极并对微阵列电极的微电极进行编号;
将糖溶液泵入检测腔;
实时记录所述微阵列电极的多路通道测试信号,并根据检测信号描绘该糖溶液的乳酸菌代谢曲线。
12.根据权利要求11所述的糖类乳酸菌代谢分析检测的方法,其特征在于:缓冲液中包含1.0×107CFU/ml的乳酸菌;所述糖溶液的泵送速度为300-600mL/h,检测温度为37℃-38℃。
13.根据权利要求11所述的糖类乳酸菌代谢分析检测的方法,其特征在于:没有味觉细胞耦合的电极输出的信号作为测试基线,有味觉细胞耦合电极输出的测试信号。
14.根据权利要求12所述的糖类乳酸菌代谢分析检测的方法,其特征在于:不同糖溶液注入的最小时间间隔为5分钟;在两种糖溶液检测之间,用新鲜标准灌流液冲洗测试腔,再用所述缓冲液冲洗测试腔。
15.根据权利要求11所述的糖类乳酸菌代谢分析检测的方法,其特征在于:所述糖溶液包括75mmol/L蔗糖溶液,以及含有三氯蔗糖、赤藓糖醇、麦芽糖醇和维生素C的、甜度与75mmol/L蔗糖溶液一致的且酸度为pH3.0-4.0的混合溶液两种糖溶液。
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