一种β-内酰胺类抗生素的酶热检测方法  

A Novel Method to Quantitate β-lactam Antibiotics Using an Enzymatic Thermistor Biosensor

作　　者：柳世超 李彬春[2] 李深伟[3] 吴长新 谢斌[4] 孟庆来 LIU Shichao;LI Binchun;LI Shenwei;WU Changxin;XIE Bin;MENG Qinglai(Institutes of Biomedical Sciences,Shanxi University,Taiyuan 030006,China;Institute of Biotechnology,Shanxi University,Taiyuan 030006,China;Shanghai International Travel Healthcare Center,Shanghai 200335,China;Department of Applied and Pure Biochemistry,Lund University,Lund,SE-2100,Sweden)

机构地区：[1]山西大学生物医学研究院,山西太原030006 [2]山西大学生物技术研究所,山西太原030006 [3]上海国际旅行卫生保健中心,上海200335 [4]隆德大学纯粹与应用生物化学系,瑞典隆德SE-2100

出　　处：《山西大学学报（自然科学版）》2021年第2期335-341,共7页Journal of Shanxi University(Natural Science Edition)

基　　金：国家重点研发计划“高端试剂可溯源量值定值技术及质量评价方法研究”(2019YFF0216500)。

摘　　要：组装有青霉素酶酶柱的酶热传感器(以下简称"青霉素酶酶热传感器")可应用于青霉素类抗生素血药浓度的快速检测及牛奶中外源添加的青霉素酶(解抗剂)的现场快速检测,但由于该传感器对头孢霉素类及碳青霉烯类抗生素检测活性差而限制了其广泛应用。近期出现的一种碳青霉烯酶"新德里金属β-内酰胺酶-1,NDM-1"则具有水解几乎所有β-内酰胺类抗生素的活性。文章制备了固相有NDM-1酶的酶柱,并以该酶柱组装酶热传感器成为NDM-1酶热传感器。NDM-1酶热传感器对哌拉西林、头孢曲松和美罗培南均表现出高水平的检测活性,且检测活性依赖Zn2+存在。在抗生素浓度为31.25 mg/L~1 000 mg/L浓度范围内,NDM-1酶热传感器对青霉素G、哌拉西林、头孢唑啉、头孢曲松、头孢吡肟及美罗培南的反应活性随抗生素浓度增加而明显增加,且显著线性相关。在pH值为6.0~8.0的范围内,NDM-1酶热传感器对青霉素G、哌拉西林、头孢唑啉、头孢曲松及头孢吡肟表现出最优反应活性时对应的pH值为6.5;而对美罗培南表现出最优反应活性时对应的pH值为8.0。研究表明应用NDM-1酶热传感器成功建立了一种可对青霉素类、头孢霉素类及碳青霉烯类三大类β-内酰胺抗生素进行高效定量分析的方法。The penicillinase thermistor biosensor(Penicillinase sensor) was developed for the rapid monitoring of blood penem antibiotics concentration and rapid identification of extraneous penicillinase in milk on site.However, the wide application of the penicillinase thermistor biosensor was limited due to its intrinsic poor activity to hydrolyze cephem and carbapenem antibiotics.The recently identified carbapenemase New Delhi metallo-beta-lactamase 1(NDM-1) is able to hydrolyze all commercially available β-lactam antibiotics in high efficacy.We coupled the NDM-1 and the enzymatic thermistor biosensor to develop a NDM-1 thermistor biosensor(NDM-1 sensor) by the installment of the enzymatic thermistor with an enzyme column loaded with NDM-1 conjugated CPG beads.The NDM-1 sensor shows high response activity to Piperacillin(PIP),Ceftriaxone(CTRX), and Meropenem(MEM), and the response activity of the NDM-1 sensor to these three β-lactam antibiotics are all Zn2+ dependent.Moreover, the response activity of the NDM-1 sensor to Penicillin G(P), PIP, Cefazolin(CZO), CTRX, Cefepime(FEP) and MEM all linearly correlated with antibiotic concentration from 31.25 to 1 000 mg/L.Within pH from 6.0 to 8.0, the optimal response activity of the NDM-1 sensor to P,PIP, CZO, CTRX and FEP are found at pH 6.5, while the optimal response activity of the NDM-1 sensor to MEM is found at pH8.0.These data indicate that the featured activity of NDM-1 was well maintained after conjugation on CPG beads, and NDM-1 sensor is capable to quantitate three classes of β-lactam antibiotics including penem, cephem and carbapenem within a wide concentration range.

关 键 词：Β-内酰胺抗生素 酶热传感器 新德里金属β-内酰胺酶-1 抗生素耐药 血药浓度 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]