Rapid Detection of Bifidobacterium Bifidum in Feces Sample by Highly
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ORIGINAL RESEARCH published: 07 July 2020 doi: 10.3389/fchem.2020.00548 Rapid Detection of Bifidobacterium bifidum in Feces Sample by Highly Sensitive Quartz Crystal Microbalance Immunosensor Kaijian Hou 1†, Pingsen Zhao 2†, Yongru Chen 3†, Guiping Li 4, Yu Lin 5, Danjie Chen 6, Dan Zhu 1, Zezhen Wu 1, Danchun Lian 7, Xiaojun Huang 7 and Jilin Li 7* 1 Department of Endocrine and Metabolic Diseases, Longhu Hospital, The First Affiliated Hospital of Shantou University Medical College, Shantou, China, 2 Department of Laboratory Medicine, Yuebei People’s Hospital, Shantou University Medical College, Shaoguan, China, 3 Department of Emergency Intensive Care Unit (EICU), The First Affiliated Hospital of Shantou University Medical College, Shantou, China, 4 Department of Endocrine and Metabolic Diseases, The Third People’s Hospital of Huizhou, Huizhou, China, 5 Department of Endocrinology, Jieyang People’s Hospital, Jieyang, China, 6 Department of Endocrinology, Puning People’s Hospital, Puning, China, 7 Department of Cardiology, The Second Affiliated Hospital of Shantou University Medical College, Shantou, China Edited by: Hassan Karimi-Maleh, In this work, a quartz crystal microbalance (QCM) sensor has been fabricated using University of Electronic Science and immunoassay for sensitive determination of Bifidobacterium bifidum. Au nanoparticle Technology of China, China has been used for amplifying sandwich assays. The proposed immunosensor exhibited Reviewed by: a linear detection range between 103 and 105 CFU/mL with a limit of detection of 2.1 Somaye Cheraghi, 2 Shahid Bahonar University of × 10 CFU/mL. The proposed immunosensor exhibited good selectivity for B. bifidum Kerman, Iran sensing with low cross reactivity for other foodborne pathogens such as Lactobacillus Vahid Arabali, Islamic Azad University Sari acidophilus, Listeria monocytogenes, and Escherichia coli. In addition, the proposed Branch, Iran immunosensor has been successfully used for B. bifidum detection in feces samples and *Correspondence: food samples. The frequency decreases of 12, 17, and 10 Hz were observed from the Jilin Li milk samples consisting of the mixtures of L. acidophilus, L. monocytogenes, and E. coli. [email protected] The frequency decreases of 8, 15, and 7 Hz were observed from the feces samples † These authors have contributed consisting of the mixtures of L. acidophilus, L. monocytogenes, and E. coli. equally to this work Keywords: quartz crystal microbalance sensor, Bifidobacterium bifidum, immunosensor, Au nanoparticle, feces Specialty section: sample This article was submitted to Analytical Chemistry, a section of the journal INTRODUCTION Frontiers in Chemistry Received: 28 April 2020 Under normal circumstances, the intestinal microorganisms in the human body form a relatively Accepted: 27 May 2020 balanced state. Once the balance is damaged, it will lead to the imbalance of intestinal flora (Horie Published: 07 July 2020 et al., 2017; Xue et al., 2018). Some intestinal microorganisms, such as Clostridium perfringens, Citation: overproduce in the intestinal tract and produce harmful substances such as ammonia, amines, Hou K, Zhao P, Chen Y, Li G, Lin Y, hydrogen sulfide, manure, indole, nitrite, and bacterial toxins, which will further affect the health Chen D, Zhu D, Wu Z, Lian D, of the body (Ashida et al., 2018; Duranti et al., 2019). Bifidobacterium bifidum is an important Huang X and Li J (2020) Rapid intestinal beneficial microorganism. B. bifidum, as a physiological beneficial bacterium, has many Detection of Bifidobacterium bifidum in Feces Sample by Highly Sensitive important physiological functions for human health, such as biological barrier, anti-tumor, immune Quartz Crystal Microbalance enhancement, improvement of gastrointestinal function, and anti-aging (Wang et al., 2016; Mauras Immunosensor. Front. Chem. 8:548. et al., 2018; Din et al., 2020). B. bifidum can inhibit the growth of harmful bacteria, resist the doi: 10.3389/fchem.2020.00548 infection of pathogenic bacteria, and synthesize vitamins needed by human body. It can promote Frontiers in Chemistry | www.frontiersin.org 1 July 2020 | Volume 8 | Article 548 Hou et al. Immunosensor for Bifidobacterium bifidum Detection the absorption of minerals, produce organic acids such as acetic Ltd (Wuhan, China). Mouse IgG, 11-mercaptoundecanoic acid acid, propionic acid, butyric acid, and lactic acid to stimulate (11-MUDA), bovine serum albumin (BSA), and ethanolamine intestinal peristalsis and promote defecation. It can prevent hydrochloride were purchased from Linc-Bio Science Co., Ltd constipation, purify the intestinal environment, decompose (Shanghai, China). Au colloidal (AuNPs) with 20 nm was carcinogens, stimulate the human immune system, and improve purchased from Shenzhen Nano Tech Co., Ltd (Shenzhen, the disease resistance (Gomi et al., 2018; Bondue et al., 2019; China). All other common regents were supplied by Sinopharm Speciale et al., 2019). It is of crucial importance to identify Chemical Reagent Co., Ltd (Shanghai, China) and used B. bifidum either in food and excreta. Although the normal without further purification. A QCA922 quartz crystal analyzer microbiological isolation methods have been widely used for (Princeton, USA) has been used for sensing with an Au coated identifying bacterial strains, they are time-consuming (Yang et al., chips. Bifidobacterium bifidum (B. bifidum), L. acidophilus, L. 2017; Cheng et al., 2018; Faraki et al., 2020). Therefore, the monocytogenes, and E. coli strains were purchased from American development of a fast method for B. bifidum identification is very Type Culture Collection (ATCC, Manassas, VA). MRS (de Man, essential for sample scanning. Rogosa, and Sharpe) and Bifidus Selective Medium (BSM) were The biosensor first immobilizes the bioactive components used as growth media. (enzyme, antibody, tissue, cell) on the transducer. When the target analyte is recognized by the immobilized bioactive Sensor Chip Preparation components, the biochemical reaction can be immediately Piranha solution has been used for removing any impurities converted into a quantifiable electrical signal through the on the QCM Au chip. Then, the Au chip was immersed into transducer (Fu et al., 2015; Huertas et al., 2019; Alamgholiloo 20 mM thiol (11-MUDA, prepared using ethanol) overnight for et al., 2020; Fouladgar et al., 2020; Karimi-Maleh et al., 2020). fabricating carboxy-terminated thiol layer on the Au surface. Since the rise of biosensor in the late 1960s, after nearly half a After rinse by ethanol and water, the Au chip was used for century of development, biosensor has become a comprehensive baseline correction under 10 mM PBS (pH 7.4). A mixture and interdisciplinary field, which is used in food safety testing, solution of EDC (0.4M)-NHS (0.1M) at ratio of 1:1 was environmental testing, and clinical diagnosis. QCM is a new then used for sensor activation. Then, different concentrations type of micro mass sensor based on quartz crystal resonance, of polyclonal or mouse monoclonal antibodies against B. which was developed in 1960s. According to the piezoelectric bifidum were immobilized on the Au chip. Then, capture and effect of quartz crystal, the resonance frequency of quartz control were carried out by injected either polyclonal or mouse crystal will change with the mass of adsorbed material, and monoclonal antibodies and mouse IgG antibody. Then, BSA they are in a positive proportion (Bearzotti et al., 2017; Speller (50 µg/mL) and ethanolamine (1 M) were used for blocking and et al., 2017; Wang A. et al., 2017; Ayankojo et al., 2018). capping the sensors. QCM immunosensor is a specific biosensor combining the high sensitivity of quartz crystal and the high specificity of immune Detection of B. bifidum response (Bearzotti et al., 2017; Speller et al., 2017; Wang L. Direct detection of B. bifidum was carried out by the injection of et al., 2017; Ayankojo et al., 2018; Zhang et al., 2018). At different concentrations of B. bifidum cells suspension prepared present, QCM immunosensor has been widely used in clinical in PBS over above-mentioned polyclonal, mouse monoclonal, testing, food hygiene, environmental testing, as well as other and mouse IgG antibodies fabricated sensor. The detection was fields of chemical analysis, and biological analysis. The detection carried out by comparing either B. bifidum captured polyclonal of bacteria by QCM is a new and attractive Research Topic in or mouse monoclonal antibodies with the control group. electronic informatics and medicine (Muckley et al., 2016; Tai The sandwich assay with antibody-AuNPs conjugation et al., 2016; Chen et al., 2018; Ding et al., 2018; Lal and Tiwari, was prepared according to previous methods with some 2018; Temel et al., 2019). modifications (Uludag and Tothill, 2012). Typically, 0.1mL of In this work, a QCM immunosensor has been developed polyclonal or mouse monoclonal antibodies was added into for sensitive determination of B. bifidum. Monoclonal and 0.5 mL of AuNPs suspension with 2 h slow stirring. Then, 0.1 mL polyclonal antibody have been used with the signal amplifying of BSA (10%) was introduced to the mixture with 2 h slow based on the Au nanoparticle. The proposed immunosensor stirring. A centrifugation process was carried out to remove exhibited a wide linear detection range with a low detection the excess of BSA and AuNPs. The detection process of using of limit. The sensitivity was enhanced when the antibody- antibody-AuNPs conjugation was similar to the direct detection conjugated Au nanoparticle.