br Materials and methods br Results and discussion br Conclu

Materials and methods
Results and discussion
Conclusion Amino-functionalized mesoporous TiO2-NH2 microparticles were used to immobilize ADA, followed by GLU cross-linking. The mesopores provided a natural microenvironment with sufficient room for the enzyme to efficiently perform its catalytic function. The GLU on the particle interface acted as tongs to prevent enzyme leaching and inhibit the loss of enzyme activity. Thus, ADA-loading efficiency and recycling and storage stability were remarkably improved. As a novel and efficient carrier for enzyme immobilization, the composite developed herein may have utility in biomedical, biosensor, and other biotechnology fields. Our technique for enzyme immobilization can stabilize the enzyme via multipoint covalent attachments.
Introduction Adenosine deaminase (ADA), a key hydrolytic enzyme for purine metabolism, can catalyze the conversion of adenosine (AD) to inosine by the removal of an amino group [1]. It plays a key role in the differentiation and maturation of the lymphoid system. Moreover, accumulating evidence indicate that the dysfunction of ADA in human body is closely related to a number of important diseases, such as tuberculosis, sarcoidosis, cancer and severe combined immunodeficiency (SCID) [2], [3]. The significance of ADA in pathology makes it an important target for drug development and diseases detection. Traditional methods including the measuring ammonia produced [4], high-performance liquid chromatography (HPLC) [5], and colorimetric assay [6] have been described to be effective for monitoring ADA activity. Although each method has its advantages, many reported techniques still suffer the drawbacks, such as time-intensive, laborious, and low sensitivity. Thus, the search for simple and sensitive assay of ADA is ongoing. With rapid development in the field of DNA biotechnology, the oligonucleotides have emerged as attractive recognition units for monitoring enzymes activities. A series of DNA-based probes have been developed for sensitive activity assays of various enzymes, such as DNA methyltransferases [7], endonucleases [8], RNase H [9] and sik inhibitor sale [10]. In recent years, the aptamers have received tremendous attention in sensing applications because of their relative ease of isolation and modification, high affinity and specificity toward targets, and resistance against denaturation [11]. Up to now, several methods based on the use of aptamers as recognition units have been developed for the quantitative determination of ADA activity. These include the electrochemical aptasensor [12], colorimetric aptasensor [13] and fluorescence sensor [14]. Although these techniques can be quite powerful, a simpler and more sensitive method for ADA detection is still required. The carbon nanomaterials have a significant role to play in new developments in each of the biosensor size domains. This significance arises as nanomaterials can help address some of the key issues in the development of all biosensors [15]. For example, a series of graphene oxide-based aptasensors have been developed for diverse biomolecules detection [16], [17], [18], the carbon nanoparticles and carbon nanodots have also extensively been used to develop biosensors [19], [20]. Recently, carbon nanotubes (CNTs) have been extensively studied due to their unique optoelectronic properties and excellent biocompatibility [19]. Specifically, the extraordinary fluorescence quenching property of CNTs has been employed to develop nanosensors for diverse biomolecules in homogeneous solution. For example, the CNTs has used as a biosensing platform for the detection of DNA based on the quenching effect on the dye-labeled DNA probe [21]. Similarly, the CNTs-based aptasensors were also used for sensitive detection of various biomolecules [22], [23]. However, to the best of our knowledge, no study has been reported the use of CNTs for homogeneous assay of ADA assay. In the present work, we developed a sensitive and selective fluorescent aptasensor based on multi-walled carbon nanotubes (MWCNTs) using AD as the substrate for ADA activity detection and inhibitor screening. This aptasensor relies on the high fluorescence quenching property of MWCNTs and the different interaction ability of aptamer, AD-aptamer complex with MWCNTs. Compared with the traditional fluorescence resonance energy transfer (FRET)-based aptasensor [14], the proposed assay only requires the labeling of the oligonucleotide probe with one dye, which is very simple and cost-effective. Moreover, this aptasensor exhibits high sensitivity and specificity toward ADA over other non-specific enzymes, with a detection limit of 0.002U/mL for ADA. In addition, the suitability of this MWCNT-based fluorescence aptasensor for ADA inhibitor screening from TCM has also been demonstrated.