Current Biotechnology

Progress on Physicochemical Properties and Applications of Functional Nucleic Acid DNA Hydrogels
MA Xuan¹, ZHANG Yangzi¹, XU Wentao^1,2*
2019, 9(6): 545-553. DOI: 10.19586/j.2095-2341.2019.0046
Abstract ( ) PDF (4033KB)( )

Since DNA was developed as a nanoscale self-assembly material, it has connected the two major fields of life science and material science with its adjustable multifunctionality, convenient programmability, accurate molecular recognition capability, high versatility, superior biocompatibility and biodegradability. Benefiting from the structural properties of DNA nanomaterials, functional nucleic acid DNA hydrogels formed by cross-linking and self-assembly using functional nucleic acids as building blocks have become a research hotspot in the field of new materials. Based on this situation, the main physicochemical properties of functional nucleic acid DNA hydrogels were summarized. In the meanwhile, the application progress of functional nucleic acid DNA hydrogels in the fields of biomedicine, molecular detection and environmental engineering such as drug delivery and targeted therapy, biosensing, construction of three-dimensional tissues, etc. in recent years was further reviewed. Finally, the key points and directions that should be considered while designing and constructing functional nucleic acid DNA hydrogels were summarized from the perspectives of life science and material science, in order to assist the research and application of DNA hydrogels in multidisciplinary fields.

Preparation and Assembly of Functional Nucleic Acid DNA Hydrogels
MA Xuan¹, ZHANG Yangzi¹, XU Wentao^1,2*
2019, 9(6): 554-562. DOI: 10.19586/j.2095-2341.2019.0051
Abstract ( ) PDF (2928KB)( )

Functional nucleic acid DNA hydrogel is a kind of new flexible material which is self-assembled by chemical reaction or physical entanglement using DNA as building unit. And its building unit contains one or more specific sequences which are capable of forming functional nucleic acid. Functional nucleic acid is a kind of specific nucleic acid structure combined by base modification and interaction force between DNA molecules, including nucleic acid aptamer, DNAzymes, G-quadruplex (G4) and i-motif structures, etc. Traditionally, long DNA strands with high concentration are necessary conditions for the preparation of DNA hydrogels. But the introduction of nucleic acid amplification methods provides new possibilities for the assembly of DNA hydrogels. Therefore, various functional nucleic acids commonly used in the preparation of DNA hydrogels and methods of extraction, synthesis and amplification of nucleic acids were introduced in detail. On this basis, the preparation methods of functional nucleic acid DNA hydrogels assembled by chemical or physical crosslinking were reviewed. Finally, the challenges and potential development directions of DNA nanomaterial assembly were proposed in order to provide references for the development of functional nucleic acid DNA hydrogels with high efficiency assembly.

Special Secondary Structures of DNA and Their Application Progress
DU Zaihui¹, LUO Yunbo¹, ZHU Longjiao¹, XU Wentao^1,2*
2019, 9(6): 563-570. DOI: 10.19586/j.2095-2341.2019.0086
Abstract ( ) PDF (5173KB)( )

Nucleic acid plays an important role in the genetic process of life. Its special secondary structures of DNA not only contain genetic information, but also play a specific physiological function in vivo and be used as a component of biosensors in vitro. At present, the special secondary structures of DNA mainly include hairpin, cruciform, double helix, triplex, G-quadruplex, G-triplex and i-motif. The special secondary structures of DNA have been extensively studied both in vitro and in vivo. Therefore, based on the existing research results, the development history, structural composition, special functions and applications in biosensing, nanometer materials, in vivo detection and other fields of G-quadruplex, G-triplex and i-motif which belong to the special secondary structures of DNA were summarized. Finally, the existing problems and deficiencies in the research of DNA special secondary structures were analyzed, and their future research direction was prospected, so as to provide theoretical support for the application of DNA special secondary structure in biosensing, molecular medicine and other fields.

A New RNA Editing Technology: CRISPR-Cas13
LIU Yanjun, CHEN Zhisheng*
2019, 9(6): 571-578. DOI: 10.19586/j.2095-2341.2019.0054
Abstract ( ) PDF (5607KB)( )

CRISPR-Cas13 system is a fast, efficient and accurate RNA editing tool, which has characters of easy to design, simple structure, convenient operation and strong specificity. This paper summarized the position of CRISPR-Cas13 in CRISPR classification system, the structural basis and mechanism of CRISPR-Cas13, the comparison with other RNA level adjustment methods and the current application prospect, which was expected to provide reference for related research.

Research Progress on CRISPR-Cas Mediated Biosensors
LI Kai^1,2, LUO Yunbo^1,2, XU Wentao^1,2*
2019, 9(6): 579-591. DOI: 10.19586/j.2095-2341.2019.0079
Abstract ( ) PDF (15118KB)( )

Clustered regular interspaced short palindromic repeats (CRISPR) system is a unique immune defense mechanism that widely existed in bacteria. It can effectively bind exogenous nucleic acid molecules when combined with special Cas protein, then cause fragmentation and further degradation of exogenous gene. Due to the special recognition property of the CRISPR-Cas system, it can be used for targeted editing of genomes through directional modification in vitro. The special targeting mechanism of CRISPR-Cas system provides a new concept for the development of nucleic acid-based biosensors. More and more researchers began establishing unique logic systems based on the nature of different kinds of Cas proteins, to achieve accurately the identification of target substances. Biosensors based on CRISPR technology had also opened up the application in other fields. This paper introduced the origin, mechanism and scientific classification of CRISPR-Cas system and classified the biosensors according to different mechanisms and identify substrates. Finally, we prospected for the application of efficient CRISPR based on biosensors.

Research Progress of DNA Sensors Based on Surface Plasmon Resonance and Electrochemical Combination
ZHANG Yukun1,2, AN Na2, LIU Weixiao2, WAN Yusong2, JIN Wujun2, LI Liang2*, ZHANG Xiao1*
2019, 9(6): 592-598. DOI: 10.19586/j.2095-2341.2019.0067
Abstract ( ) PDF (1304KB)( )

Surface plasmon resonance (SPR) is designed to detect the change of refractive index near the surface. It is characterized by unmarked, real-time, sensitive and fast. It is often used to study molecular interactions, including kinetics, efficiency constant and macromolecular conformational change and so on. Electrochemical (electrochemical, EC) technology is a mature technology for qualitative and quantitative study of electron transfer, redox and interfacial adsorption. It has the advantages of simplicity, low cost and miniaturization of equipment. The existing DNA hybridization techniques, such as optical, electrochemical or piezoelectric transduction techniques, mainly focus on improving the selectivity and sensitivity of DNA hybridization detection system. In the area of DNA analysis, the traditional SPR is limited in the application of ultra-sensitive detection because it is unable to measure the minimal change of refractive index. Therefore, with the development of nanomaterials and the rapid development of hyphenated technology, the research of biosensors combined with SPR and EC has become the focus with more and more attention. In recent years, there have been few report on the combination of SPR and EC in the detection of DNA. Therefore, this paper briefly introduced the technical principle, hyphenated methods and application progress of SPR and EC detection of DNA, which was expected to provide reference for the research of DNA sensors based on surface plasmon resonance and electrochemical combination.

Progress on Biosensors Based on Loop-mediated Isothermal Amplification
LIN Shenghao1, DU Zaihui1, ZHANG Xiujie2, HUANG Kunlun1,3, LIU Qingliang4, XU Wentao1,3*
2019, 9(6): 599-610. DOI: 10.19586/j.2095-2341.2019.0069
Abstract ( ) PDF (1187KB)( )

Loop-mediated isothermal amplification (LAMP) is characterized by rapid, isothermal, high specificity, and simple operation, etc. Therefore, biosensors based on the LAMP principle have been widely developed and utilized in recent years. Based on this situation, LAMP was introduced, including primer design principle, composition and function of amplification system, specific amplification process, advantages and disadvantages. Then, the colorimetric biosensors, fluorescent biosensors, electrochemical biosensors and other kinds of biosensors based on LAMP were mainly introduced, and the characteristics of various kinds of biosensors were summarized and compared. Finally, the shortcomings of LAMP biosensors were analyzed, and the development direction of LAMP biosensors was prospected, so as to provide references for the research and development of low-cost, highly sensitive, automated and miniaturized LAMP biosensors in the future.

Colorimetric Sensors for Cd²⁺ and Pb²⁺ Detection Based on the Peroxidase Activity of Fe₃O₄
YANG Wenping^1,2, WU Yuangen^1*
2019, 9(6): 611-619. DOI: 10.19586/j.2095-2341.2019.0093
Abstract ( ) PDF (7105KB)( )

Excessive heavy metal residues is one of the major food safety issues. To quickly and accurately determine the amount of heavy metals, this paper reported a colorimetric sensor for the quantification of Cd²⁺ and Pb²⁺. The peroxidase activity of Fe₃O₄ was used as the signal output tool and aptamer as the target specific recognition element. First, the detection method was determined by the feasibility analysis and the condition optimization, and the sensor performance was evaluated by sensitivity and specificity experiments. Under the optimum conditions, the sensor has a high selectivity over other competitive ions with the detection limits as low as 0.108 μmol/L for Cd²⁺ and 0.117 μmol/L for Pb²⁺. The method has the advantages of low cost, fast and simple detection process, visible to the naked eye, and the quantitative analysis can be realized by hand-held photometer. The establishment of the sensor would make it possible to detect heavy metals in real time and quickly, and provide a strong guarantee for food safety detection in China.

Research Progress on the Recognition Mechanism of Gas Biosensors
SU Yuan¹, LI Shuting¹, XU Wentao^1,2*
2019, 9(6): 620-626. DOI: 10.19586/j.2095-2341.2019.0049
Abstract ( ) PDF (2278KB)( )

Gas biosensors are sensors based on biometrics, biotransformation, and gas signal output. It constructs a gas production system with “enzyme”, using gas concentration meter, barometer, pH meter to detect gas concentration value, barometric pressure value, pH value, and observe the color change by visual observation to establish a relationship between concentration, pressure, pH, color and target material to achieve rapid, accurate and sensitive quantitative and qualitative detection of target substances. This paper introduced the properties of gas biosensors, summarized the classification of gas biosensors, and also summarized the signal recognition principle and signal output mode of gas biosensors. The sensing materials and construction methods of gas biosensors were prospected.

Advances on the Application of Gas Biosensors
SU Yuan¹, LI Shuting¹, XU Wentao^1,2*
2019, 9(6): 627-632. DOI: 10.19586/j.2095-2341.2019.0048
Abstract ( ) PDF (2064KB)( )

Gas biosensors are sensors based on biometrics, biotransformation, and gas signal output. In recent years, gas biosensors have been used in the detection of target substances such as biomarkers, cells, and proteins, because of their simplicity, sensitivity, and specificity. This paper briefly introduced the nature and classification of gas biosensors, and described the principles and applications of protease-mediated gas biosensors, nuclease-mediated gas biosensors, mimic enzyme-mediated gas biosensors and other gas biosensors. The detection methods and application prospects of gas biosensors were prospected, which provided constructive reference for the research of gas biosensors.

Progress on Biosensor Based on Malachite Green Aptamer
FENG Min1, DU Ning1, ZHANG Yangzi2, LI Xiangyang3, XU Wentao2*, LIU Haiyan1*
2019, 9(6): 633-640. DOI: 10.19586/j.2095-2341.2019.0095
Abstract ( ) PDF (930KB)( )

As a fluorescent probe without labeling, fluorescent aptamer has many potential advantages and has been applied to detect a variety of target substances (such as ATP, RNA), which is currently a hot spot in the field of aptamer research. Malachite green aptamer (MGA) belongs to fluorescent aptamer, which can form binding pockets through ligand-induced folding, thus promoting the luminescence of malachite green (MG). At present, there are few kinds of MGA that have been screened out. Therefore, the characteristics of known MG RNA aptamers and their allosteric bodies, MG DNA aptamers were mainly introduced, as well as the factors affecting the fluorescence intensity of MG-MGA complexes. At the same time, the main MG derivatives and copolymers were also summarized. Finally, the applications of MGA in biosensing and fluorescence imaging were reviewed, and the development direction of MGA was prospected, so as to provide guidance for the application of MGA in biological detection and bioimaging, etc.

A Colorimetric Biosensor Based on G-quadruplex for Detection of Sheep-derived Components
WANG Xinyi1, LIU Bang1, SHEN Yang1, XU Wentao2, ZHOU Xiang1*
2019, 9(6): 641-646. DOI: 10.19586/j.2095-2341.2019.0104
Abstract ( ) PDF (3625KB)( )

In recent years, the phenomenon of meat adulteration has become a hot topic in society. Identification of animal-derived components in livestock products is one of the important strategies to fight against meat adulteration. Based on this situation, a colorimetric biosensor based on G-quadruplex was established to identify sheep-derived components in meat products by using asymmetric PCR (As-PCR) and combining with the functional characteristics of G-quadruplex. Using sheep genomic DNA as template, and adding a reverse complementary sequence of G-quadruplex to the 5′ end of the restrictive forward primer, a large amount of single strand DNA (ssDNA) containing G-quadruplex sequence was amplified by As-PCR, and then the generated ssDNA combined with hemin under the action of K+ to form the G-quadruplex-hemin complex with DNAzyme activity. The compound could catalyze the reaction of H2O2 and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS2-) to generate a green reaction product, and the result could be visually observed without conventional detection steps, such as electrophoresis, sequencing, etc. By optimizing the reaction conditions and system of As-PCR, the biosensor could realize accurate and rapid identification of sheep-derived components in meat products, and it could stably detect the minimum mass fraction of sheep-derived components in mixed meat samples to be 5%. The colorimetric biosensor based on G-quadruplex established by the research had the advantages of simplicity, sensitivity, visualization, low cost, etc. And it could be used for detecting animal-derived components in meat products, and it provided strong technical support for the safety detection of meat products.

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