CN116642877A - 一种噬菌体生物发光阵列细菌检测芯片及其制备方法和应用 - Google Patents
一种噬菌体生物发光阵列细菌检测芯片及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种噬菌体生物发光阵列细菌检测芯片及其制备方法和应用,属于食品中微生物含量检测技术领域。本发明利用聚二烯丙基二甲基氯化铵、聚乳酸和LB培养基制备噬菌体生物发光阵列细菌检测芯片基底,顺次进行细菌修饰、噬菌体修饰和发光修饰,得到噬菌体生物发光阵列细菌检测芯片。其中LB培养基吸引细菌在反应池表面吸附生长;表面带有正电基团的高分子物质聚二烯丙基二甲基氯化铵将头部带有负电的噬菌体牢固地吸附在反应池的表面。本发明制备的芯片灵敏度高、准确性高、特异性强,对致毒菌株药敏测试时间缩短在2h内,且制备工艺简单,无需采用大型设备,反应条件温和、成本低、安全系数高。
Description
技术领域
本发明涉及食品中微生物含量检测技术领域,尤其涉及一种噬菌体生物发光阵列细菌检测芯片及其制备方法和应用。
背景技术
由微生物病原体引起的食源性疾病的爆发在全球范围内非常普遍。而且食品表面显然存在不止一种细菌,且细菌浓度会因为环境原因极度复杂多变。而现有的微生物含量检测技术如直接计数法、比浊法、活菌计数法、测定重量法、测定细胞总氮量或总碳量法以及颜色改变单位法中,直接计数法、活菌计数法、测定重量法在细菌数目测定中有明显、巨大且难以消除的系统误差;比浊法检测限过高,对浓度较低的细菌溶液难以检测;颜色改变单位法、测定细胞总氮量或总碳量法无法特异性检测混合细菌溶液中单种细菌的浓度系数。这些传统微生物检测技术仅仅只能满足细菌总量的初步估算,难以给出具体的细菌浓度数据,且待测细菌浓度的检测极易受到其他细菌的干扰。因此,在进行单种细菌浓度测定的定量分析时,提供一种高精度、易操作、抗干扰能力强、特异性高、检测范围广的微生物检测技术势在必行。
光学生物传感器最近被认为是现有传统病原体检测方法的有吸引力的替代品。光学生物传感器可以感知微生物与分析物的相互作用,并根据测量参与该过程的光子,将观察到的光学信号与微生物种群的浓度相关联。光学检测主要包括测量发光、荧光发射或吸收率。三磷酸腺苷(ATP)生物发光被认为是一种非常有效的生物传感器,是存在于各种微生物中的主要生物能源,反映了活微生物的存在。可进行敏感、无损和实时检测。
虫荧光素和部分虫荧光素酶的溶液在无能量物质三磷酸甘油酸(ATP)支持的环境中不会发生反应产生生物发光现象,但是在遇到ATP后,会断键释放出一个磷酸基团并为虫荧光素酶反应虫荧光素提供能量。虫荧光素在被虫荧光素酶降解后将会释放出光,这就是生物发光检测的基本原理。
当今食品检测行业具有广阔的前景,但现有的技术面临着灵敏度低、耗时、步骤繁琐、准确率低、易受干扰、检测范围窄、精度低的问题。因此,发展一种用于食品中微生物含量检测的灵敏度高、准确性高、特异性强、测试时间短、操作简便、成本低、检测范围广的噬菌体生物发光阵列细菌检测芯片成为本领域亟需。
发明内容
本发明的目的在于提供一种噬菌体生物发光阵列细菌检测芯片及其制备方法和应用。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种噬菌体生物发光阵列细菌检测芯片的制备方法,其特征在于,包括如下步骤:
(1)将聚二烯丙基二甲基氯化铵、聚乳酸和LB培养基的混合液,加入到芯片模具中,进行固化处理,得到带有若干反应池的芯片基底;
(2)将所述芯片基底与有机玻璃基座贴合固定,得到待修饰的芯片;
(3)在所述待修饰芯片的反应池中顺次进行细菌修饰、噬菌体修饰和发光修饰,最终得到噬菌体生物发光阵列细菌检测芯片。
优选的,所述芯片基底的长、宽、高分别为:30~150mm、20~100mm、5~25mm。
优选的,所述反应池为半球形,直径为6~30mm。
优选的,所述混合液中聚二烯丙基二甲基氯化铵、聚乳酸和LB培养基的质量比为0.5~1.5:50~150:5~15。
优选的,所述混合液加入到芯片模具中后,先15~35℃静置3~4h,然后进行固化处理,所述固化处理的温度为55~65℃,时间为1~2h。
优选的,所述贴合处理前,先对所述带涂层的芯片基底与有机玻璃基座进行等离子处理。
优选的,所述等离子处理的温度为15~35℃,时间为0.5~1.5min。
优选的,所述贴合固定的温度为75~85℃,时间为1~3h。
优选的,所述细菌修饰是将细菌溶液按照0.5mL/池加入到反应池中,静置修饰。
优选的,所述细菌溶液为细菌的LB溶液,所述细菌溶液的浓度在580~660nm光波长处的OD值为1.0~2.0。
优选的,所述细菌包括金黄色葡萄球菌、大肠杆菌、硝化细菌、破伤风杆菌、乳酸菌、结核杆菌、沙门氏菌、副溶血性弧菌、大肠埃希氏菌、霍乱弧菌、单核增生李斯特氏菌、布氏杆菌和痢疾杆菌中的一种。
优选的,所述细菌修饰的时间为1~5h。
优选的,所述噬菌体修饰是将噬菌体溶液注满反应池。
优选的,所述噬菌体溶液采用含有镁离子与钙离子的DPBS缓冲溶液配置得到,所述噬菌体溶液的浓度为104~107PFU/mL。
优选的,所述噬菌体修饰的温度为15~35℃,时间为20~50min。
优选的,所述噬菌体修饰还包括噬菌体增生的过程,所述噬菌体增生的温度为15~35℃,时间为8~12h。
优选的,所述发光修饰是将发光试剂注满反应池。
优选的,所述发光试剂为虫荧光素、虫荧光素酶和PBS缓冲液按照质量体积比8~12mg:0.08~0.12mg:8~12mL配置的混合液,所述PBS缓冲液的浓度为8~12mmol/L,pH值为7.2~7.6。
优选的,所述发光修饰的单次时间为1~1.5h,所述发光修饰的次数为3~5次。
优选的,所述发光修饰每次的间隔时间为10~20min。
本发明还提供了一种上述制备方法得到的噬菌体生物发光阵列细菌检测芯片。
本发明还提供了一种上述制备方法得到的芯片在制备食品中致病菌株含量检测产品中的应用。
本发明利用聚二烯丙基二甲基氯化铵、聚乳酸和LB培养基制备噬菌体生物发光阵列细菌检测芯片基底,顺次进行细菌修饰、噬菌体修饰和发光修饰,得到噬菌体生物发光阵列细菌检测芯片。其中LB培养基吸引细菌在反应池表面吸附生长;表面带有正电基团的高分子物质聚二烯丙基二甲基氯化铵将头部带有负电的噬菌体牢固地吸附在反应池的表面。
本发明通过噬菌体特异性的捕获并杀死待测细菌,导致细菌的细胞壁解离,并释放出ATP给虫荧光素酶供能后分解虫荧光素,发出亮光。
本发明制备的芯片灵敏度高、准确性高、特异性强,对致毒菌株药敏测试时间缩短在2h内。且制备工艺简单,无需采用大型设备,反应条件温和、成本低、安全系数高。
附图说明
图1为实施例1中芯片模具的AI设计图;
图2为实施例1制备的噬菌体生物发光阵列细菌检测芯片基底的俯视图;
图3为实施例1制备的噬菌体生物发光阵列细菌检测芯片基底的俯视图(与图2标注了不同的结构尺寸);
图4为实施例1制备的噬菌体生物发光阵列细菌检测芯片基底的主视图;
图5为实施例1制备的噬菌体生物发光阵列细菌检测芯片基底的左视图;
图6为实施例1所得噬菌体生物发光阵列细菌检测芯片基底与基座贴合固定示意图;
图7为实施例2噬菌体生物发光阵列细菌检测芯片针对一系列不同浓度梯度金黄葡萄球菌株检测后得到的线性回归直线。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
噬菌体生物发光阵列细菌检测芯片的制备
以金黄色葡萄球菌为例制备噬菌体生物发光阵列细菌检测芯片
(1)噬菌体生物发光阵列细菌检测芯片基底的制备:
芯片模具制备:采用AI制图,将设计好的图形(如图1所示)导入jdpaint软件,设置雕刻路径和深度为:基底的长、宽、高分别为:60mm、40mm、10mm;半球形反应池的周长为12mm,通过雕刻机进行雕刻,得到芯片模具;
芯片模具疏水处理:将芯片模具置于质量浓度为2%的十八烷基三氯硅烷甲苯溶液中疏水处理30min,处理完后用甲苯溶液洗涤3次,室温干燥2h,得到疏水处理的芯片模具;
噬菌体生物发光阵列细菌检测芯片基底的制备:将质量比为1:100:10的聚二烯丙基二甲基氯化铵、聚乳酸和LB培养基的混合液倒入经疏水处理的芯片模具中,25℃静置3.5h后在60℃下干燥1h,得到噬菌体生物发光阵列细菌检测芯片基底。该芯片基底的俯视图、主视图、左视图如图2~5所示。
其中LB培养基;称取胰化蛋白胨10g、酵母提取物5g、NaCl 10g溶于950mL蒸馏水中,用1M的NaOH调节pH值到7.0,定容至1L。121℃,20min高压灭菌,得到LB溶液,4℃储存。
将噬菌体生物发光阵列细菌检测芯片基底与长、宽、高分别为:60mm、40mm、20mm的有机玻璃板在25℃下用等离子机处理1min,之后如图6所示的位置关系置于80℃贴合2h,得到待修饰的芯片。
(2)噬菌体生物发光阵列细菌检测芯片的制备:
细菌修饰:
称取胰化蛋白胨10g、酵母提取物5g、NaCl 10g溶于950mL蒸馏水中,用1M的NaOH调节pH值到7.0,定容至1L。121℃,20min高压灭菌,得到LB溶液,4℃储存。
称取8.2g NaCl、0.2g KCl、2.9g Na2HPO4、0.2g KH2PO4以及0.5mL吐温20,加蒸馏水定容至1000mL,得到0.01M的PBST缓冲液。
将金黄色葡萄球菌与LB溶液配制成在600nm光波长下1.6OD的金黄色葡萄球菌溶液。
分别在反应池中加入2mL金黄色葡萄球菌溶液修饰3h,待细菌吸附到反应池后,用PH=7.4的0.01M PBST缓冲液洗涤3次,25℃下干燥至反应池内无液体残留。
噬菌体修饰:
称取氯化钾0.2g,磷酸单钾0.2g,氯化钠8g,七水磷酸氢二钠2.1g,氯化钙0.1g,六水合氯化镁0.1g,用二级去离子水定容至1L,得到含有钙离子和镁离子的DPBS缓冲稀释液。将其与噬菌体配置成浓度为106PFU/mL噬菌体溶液。
将噬菌体溶液注满经细菌修饰后的反应池,25℃,修饰30min。修饰结束后,在25℃下增生10h。增生结束后用PH=7.4的0.01M PBST缓冲液清洗3次,25℃下干燥至反应池内无液体残留。
发光修饰:
称取8g NaCl、0.2g KCl、1.44g Na2HPO4、0.24g KH2PO4溶于800mL蒸馏水中,用HCl调节溶液至7.4,最后加蒸馏水定容至1L,得0.01M的PBS缓冲液。
将5g虫荧光素、2g虫荧光素酶和100mLPBS缓冲液混合均匀,得到虫荧光素和虫荧光素酶混合溶液。
虫荧光素以及虫荧光素酶混合溶液注满反应池修饰4次,每次1h,每次修饰间隔15min。得到噬菌体生物发光阵列细菌检测芯片。
实施例2
以金黄色葡萄球菌为例进行食品中微生物含量检测
(1)配制浓度为102、103、104、105、106、107、108CFU/mL的金黄色葡萄球菌标准溶液,配制溶剂为无菌的质量浓度为0.90%的生理盐水。
(2)将实施例1制备的经过灭菌处理的芯片8个独立单元中,用注射器吸取步骤(1)中配置好的浓度梯度金黄色葡萄球菌株溶液各1组(共7组)以及1组无菌的质量浓度为0.90%的生理盐水各100μL,分别注射入相应反应池位置,在37℃下培养3h,3个重复。
(3)将制得的噬菌体生物发光阵列细菌检测芯片的反应池浸在pH值为7.4的PBST缓冲溶液中,浸泡时间为2h;无菌室内干燥1h,至反应池内无液体残留。
(4)将步骤(3)富集完毕金黄色葡萄球菌株的生物发光阵列细菌检测芯片置于生物发光检测仪,记录8组反应池内的相对光单元值,将注射无菌的质量浓度为0.90%的生理盐水组的相对光单元值记为(BL0),再将剩余7组反应池内的相对光单元值按次序记为(BLi),将BLi与BL0相减,分别得到相对光单元值(ΔBLi)。即不同浓度的金黄色葡萄球菌株的相对光单元值分别为BL2、BL3、BL4、BL5、BL6、BL7、BL8。根据上述不同浓度的金黄色葡萄球菌株的相对光单元值以及浓度取Log10对数,线性拟合,将金黄色葡萄球菌株的浓度为横坐标,金黄色葡萄球菌株的相对光单元值为纵坐标绘制图像,得到浓度与相对光单元值的线性回归方程,如图7所示。记录线性回归方程参数,作为参照方程。
(5)市场上随机购买的,苹果一个(278g)、香蕉一根(156g)、西梅100g、香菇菜100g、豇豆100g、韭菜100g、芹菜各100g、大白菜100g,分为8组。将每组水果蔬菜用100mL无菌的0.90%的生理盐水浸泡1h。
(6)将一块新的经过灭菌处理的芯片8个独立单元中,用注射器吸取步骤(5)中浸泡完水果蔬菜后的生理盐水100μL,注射入反应池位置,在37℃下培养3h。
(7)将制得的噬菌体生物发光阵列细菌检测芯片的反应池浸在pH值为7.4的PBST缓冲溶液中,浸泡时间为2h;无菌室内干燥1h,反应池内无液体残留。
(8)将步骤(7)干燥过后的生物发光阵列细菌检测芯片置于生物发光检测仪,记录8组反应池内的相对光单元值,将8组反应池内的相对光单元值按次序记为(BLi’),将BLi’与BL0相减,分别得到对应的相对光单元值(ΔBLi’)。即不同样本的金黄色葡萄球菌株的相对光单元值分别为BL1’、BL2’、BL3’、BL4’、BL5’、BL6’、BL7’、BL8’,找到对应相对光单元值的浓度对数值。再将查找到的金黄色葡萄球菌浓度对数值,加3后取指数10,即可得到不同品种的水果蔬菜上的金黄葡萄球菌的浓度值。
检测结果显示,苹果检测出金黄色葡萄球菌浓度为56CFU/g,香蕉中检出金黄色葡萄球菌浓度为106CFU/g,西梅中检出金黄色葡萄球菌浓度为284CFU/g,香菇菜中金黄色葡萄球菌浓度为51CFU/g,豇豆中金黄色葡萄球菌浓度为47CFU/g,韭菜中金黄色葡萄球菌浓度为46CFU/g,芹菜与大白菜中未检测出金黄色葡萄球菌。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种噬菌体生物发光阵列细菌检测芯片的制备方法,其特征在于,包括如下步骤:
(1)将聚二烯丙基二甲基氯化铵、聚乳酸和LB培养基的混合液,加入到芯片模具中,进行固化处理,得到带有若干反应池的芯片基底;
(2)将所述芯片基底与有机玻璃基座贴合固定,得到待修饰的芯片;
(3)在所述待修饰芯片的反应池中顺次进行细菌修饰、噬菌体修饰和发光修饰,最终得到噬菌体生物发光阵列细菌检测芯片。
2.根据权利要求1所述的制备方法,其特征在于,所述芯片基底的长、宽、高分别为:30~150mm、20~100mm、5~25mm;所述反应池为半球形,直径为6~30mm。
3.根据权利要求2所述制备方法,其特征在于,所述混合液中聚二烯丙基二甲基氯化铵、聚乳酸和LB培养基的质量比为0.5~1.5:50~150:5~15;
所述混合液加入到芯片模具中后,先15~35℃静置3~4h,然后进行固化处理,所述固化处理的温度为55~65℃,时间为1~2h。
4.根据权利要求3所述制备方法,其特征在于,所述贴合处理前,先对所述带涂层的芯片基底与有机玻璃基座进行等离子处理;
所述等离子处理的温度为15~35℃,时间为0.5~1.5min;
所述贴合固定的温度为75~85℃,时间为1~3h。
5.根据权利要求4所述制备方法,其特征在于,所述细菌修饰是将细菌溶液按照0.5mL/池加入到反应池中,静置修饰;
所述细菌溶液为细菌的LB溶液,所述细菌溶液的浓度在580~660nm光波长处的OD值为1.0~2.0;
所述细菌包括金黄色葡萄球菌、大肠杆菌、硝化细菌、破伤风杆菌、乳酸菌、结核杆菌、沙门氏菌、副溶血性弧菌、大肠埃希氏菌、霍乱弧菌、单核增生李斯特氏菌、布氏杆菌和痢疾杆菌中的一种;
所述细菌修饰的时间为1~5h。
6.根据权利要求5所述制备方法,其特征在于,所述噬菌体修饰是将噬菌体溶液注满反应池;
所述噬菌体溶液采用含有镁离子与钙离子的DPBS缓冲溶液配置得到,所述噬菌体溶液的浓度为104~107PFU/mL;
所述噬菌体修饰的温度为15~35℃,时间为20~50min;
所述噬菌体修饰还包括噬菌体增生的过程,所述噬菌体增生的温度为15~35℃,时间为8~12h。
7.根据权利要求6所述制备方法,其特征在于,所述发光修饰是将发光试剂注满反应池;
所述发光试剂为虫荧光素、虫荧光素酶和PBS缓冲液按照质量体积比8~12mg:0.08~0.12mg:8~12mL配置的混合液,所述PBS缓冲液的浓度为8~12mmol/L,pH值为7.2~7.6;
所述发光修饰的单次修饰时间为1~1.5h,所述发光修饰的次数为3~5次,所述发光修饰每次的间隔时间为10~20min。
8.权利要求1~7任一项所述的制备方法得到的噬菌体生物发光阵列细菌检测芯片。
9.权利要求8所述的芯片在制备食品中致病菌株含量检测产品中的应用。
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