Perspectives in app lication of biosensors for the modernization of traditional Chinese medicine

Qiang Chen,Hong-Cai Shang,Bing-Kai Han,Jun Jiao

1The Key Laboratory of Bioactive Materials,M inistry of Education,College of Life Science,Nankai University,Tianjin China.2The Key laboratory of Chinese internal medicine of MOE,BUCM,Beijing,China.

Abstract Biosensor is an instrument which is sensitive to biological material and converts its concentration into electrical signals.Organisms such as enzymes,antibodies,tissues,cells and so on can selectively identify specific substances.These molecular recognition functions can be combined with the target,such as the binding of antibodies and antigens,and the binding of enzymes to the substrate through the recognition process.Biosensor has the advantages of high specificity,high sensitivity,fast reaction speed,low cost and easy operation.It has wide application prospect in food,pharmacy,chemical industry,clinical exam ination,biomedicine,environmental monitoring and so on,especially as a new technology means,in the field of modern herbal medicine research influence.Studies have demonstrated that the biosensing technology has been applied to,traditional Chinese medicine(TCM)targets,isolation and purification of TCM,the mechanism of TCM,quality control of TCM,the active ingredients detection of TCM and other basic researches.Biosensor technology has made an important contribution to the research of modern herbal medicine,and has become a Hot-spot in future research.

Keywords:Biosensor,Traditional Chinese medicine,Purification,Active compound identification,Mechanism,Quality control

Background

Biosensing technology is a technology that uses biological substances(such as enzymes,proteins,DNA,antibodies,antigens,biofilms,m icroorganisms,cells,etc.)as recognition elements to convert biochem ical reactions into quantifiable physical and chemical signals,thus achieving the high-tech analysis of biological substances,chemical substances detection,with conventional technology irreplaceable advantages:①real-time,biological macromolecules can interact with the real-time changes in real-time monitoring analysis;②high efficiency,it can complete a basic process takes only 1-3 minutes,a large number of samples can be measured in a short time;③sensitivity,it can detect the reaction process of 10-13 mol/L concentration;④specificity,other non-specific molecules in the sample have no effect on the test results and this progress can be completed in the sample concentration is not high;⑤simplicity,there is no need to mark the interaction of macromolecules;⑥objectivity,experimental observations were recorded and analyzed by the instrument to avoid the subjective factors influence,and ensure better repeatability.Due to these unique technological advantages,biosensing technology has been widely applied to the analysis of the interaction between biomolecules,such as proteins and proteins,antigens and antibodies,DNA/RNA and binding proteins,and will be widely used in signal transduction,Protein engineering,antibody characterization,nucleic acid research.It also plays a significant role on the development of modern herbal medicine research.

Depending on the molecular recognition element,biosensors can be classified into enzyme sensors,immunosensors,nucleic acid sensors,cell sensors,tissue sensors,etc.,According to the different transducers,biosensors can be divided into electrochem ical biosensors,optical biosensors,thermal biosensors,acoustic biosensors,piezoelectric crystal biosensors and so on.According to the molecular recognition element and the analyte interacting different ways,biosensors can also be divided into affinity biosensors and catalytic biosensors.Affinity biosensors make use of the specific affinity between biological macromolecules and analytes to change the physical properties(such as mass,thickness,optical properties,electromagnetic properties,etc.)of the immobilized matrix on the transducer,so as to achieve the purpose of determining the concentration of the analyte,which detects changes in the thermodynamic equilibrium.Common affinity type biosensors include Quartz Crystal Microbalance(QCM),Surface Plasmon Resonance(SPR),and IAsys sensors.Catalytic biosensor is the use of enzyme-specific catalytic substrate or test substance on the inhibition of enzyme to achieve the detection of the substrate under test.Common catalytic biosensors are electrochem ical sensors,cell sensors,enzyme sensors,etc.It detects the kinetic effects of enzymatic reactions.

There are other methods in the study of modern herbal medicine,such as Chromatography,Spectroscopy,Mass Spectrometry,Quantum Probe,Immune detection,etc.Compared with these technologies,Biosensor technology has its special advantages which were shown in table 1.

Table 1 Com parison of biosensor technology with other technologies

App lication and influence of biosensor as a new technology in the separation and purification of modern herbal medicine

Traditional Chinese medicine has a long history and unique curative effect,which is an important part of Chinese traditional culture.With the progress of Chinese herbal medicine extraction technology,the application of Chinese herbal medicine has been greatly developed and promoted.In order to improve the curative effect of traditional Chinese medicine,it is very important to choose the appropriate method to purify the active ingredient of traditional Chinese medicine.

In recent years,with the rapid development of science and technology,the separation and purification technology is also developing.Common traditional purification techniques which we use include:High Speed Countercurrent Chromatography(HSCCC)[7],Silica Gel Column Chromatography,Macroporous Adsorption Resin,Membrane Separation Technology,Supercritical Fluid Color[8]and High Performance Liquid Chromatography[9].At present,in view of the traditional method in the lim its of the specificity and monitoring the purity,The Molecularly Imprinted Biological Sensing Technology[10]in the separation of active ingredients in Chinese medicine is becom ing more and more attractive.It will become an important technical means in the field of separation and purification of traditional Chinese medicine in the future.Molecularly imprinted polymer(M IPs)has the advantages of high efficiency,stability and simple preparation.It can be used as the identification element of biosensor form ing bioreceptor of the molecularly imprinted sensor through specific recognition and binding capacity.According to different material under test of molecularly imprinted template design,cooperating with other technologies such as Solid Phase Extract Technology,this method not only can realize the separation and purification of material,but also can meet the material concentration on separation and purification of change in the process of real-time monitoring.

As a new technology application in the field of separation and purification of traditional Chinese medicine,biosensor technology has good selectivity and affinity.It can also both improve the purity and monitor the process of separation and purification.That makes Molecularly Imprinted Biological Sensing Technology is of great significance in the study of separation and purification of Chinese medicine.

App lication of biosensors in identification and screening of active compound

Traditional Chinese medicine(TCM)is a treasure of Chinese traditional culture.It is an important task to detect and identify the active ingredients in herbal medicine and find their therapeutic targets since TCM are always made of two or more ingredients[11].Traditional analytical methods,such as cell membrane chromatography[12]and capillary electrophoresis[13],are gradually unable to meet the requirements in sensitivity,speed and specificity.The advantages of biosensor,like high efficiency,real-time detection,specificity and sensitivity,have attracted wide attention in the identification and screening of active components in TCM.

Surface plasmon resonance(SPR)biosensor is a new type of optic analysis technology,which has advantages of high specificity,label-free,low consumption of sample[14].At present,it has been widely used in the study of Chinese medical herbs.Zhang[15]established a SPR-HPLC-MS/MS online analysis system to detect and screen active ingredients of traditional Chinese medicine.11 isoflavones and 9 Astragalus saponins had been screened out from Radix Scutellariae successfully,and false positive rate and matrix effects were reduced as well through SPR coupled with HPLC technology.The carbon chain could be fixed on the SPR chip,and the carboxyl group at the end of the carbon chain had the ability to integrate with glycyrrhizic acid in the sample.Based on this principle,a SPR biosensor had been established with a low detection lim it of 20 pg/m L,which was 50 times lower than that of indirect competitive enzyme-linked immunosorbent assay(icELISA)[16].

Electrochem ical biosensors showed promising potentials in detection of small molecules in TCM and identification of counterfeit products.Lei et al[17]fabricated an electrochemical sensor with graphene modified glassy carbon electrode by using one-step reduction method to analyze the active ingredients of traditional Chinese medicine rutin.The detection lim it was 23.2nM(S/N=3).Meanwhile,this biosensor carried out quantitative and qualitative analysis of Rutin in Flos Sophorae and Compound Rutin Tablets analysis,providing a platform for analysis of active ingredients of traditional Chinese medicine.A poly(diallyldimethylammonium chloride)functionalized graphene-based electrochem ical sensor with ultra-sensitivity towards ferulic acid had been used for determination of ferulic acid in Angelica sinensis and analyzing the recovery rate of ferulic acid in urine[18].

Molecular imprinting is a technique used in molecular recognition by form ing template-shaped cavities in polymer matrices,and it has enormous potential application in the study of screening and purification of active ingredients in Chinese medicine.He Xiwen et.al[19]used aesculetin as the template molecule,acrylam ide(AA)as the functional monomer,ethylene glycol dimethacrylate(EGDMA)as crosslinking agent,and 2,2-azobisisobutyronitrile(AIBN)as the initiator to create a molecular imprinted polymer of aesculetin.Aesculetin,the active ingredients of Ash AARK,had been screened out through simply self-made chromatographic column,which provided huge potential to apply molecular imprinting in construction of biosensor for study of Chinese medicine.

App lication and influence of biosensor as a new technology in action target and drug delivery of modern herbal medicine

The action mechanism of traditional Chinese medicine is a typical multi-component,multi-target and multi-channel integrative treatment,which can produce synergistic effect among the components,and finally achieve the goal of reducing poison.However,the research on the mechanism of Chinese traditional medicine at the molecular level is not clear enough,which hinders the modernization process and international development of modern herbal medicine.Different from traditional one-component drug “single ingredient-single target”mechanism,the characteristics of multicomponent multiple targets of Chinese traditional medicine requires the research of its mechanism must pay attention to the link between the targets or composition.It is of great significance for the modernization of traditional Chinese medicine to verify the action target of traditional Chinese medicine and to elucidate the mechanism of TCM’s action on the molecular level.In recent years,with the rapid development of bioinformatics and computer science,a variety of target prediction,identification and validation methods have emerged.The research methods of multi-component multicomponent action of traditional Chinese medicine mainly include network pharmacology[20-21],computer-aided drug design[22-23],proteomics[24-25],biochip[26-27],etc.Because 90%of known drug targets are protein,also biological sensing technology as an important research method,especially its interaction between proteins provides objective auxiliary and advantage for verification at the experimental level potential targets.The use of computer and other technical means can further improve the accuracy of multi-target prediction.

As a kind of affinity type biological sensor,Quartz Crystal Microscale(QCM)is used as a biosensor to monitor quality changes.The biological sensing interface is constructed through specificity of protein identification.In the process of protein interactions,it produces a quality signal that can transfer into electrical signal.It can describe the relations in the process of protein interactions[28].

Biosensor is also an important technology in Chinese medicine carrier and delivery research.With the in-depth study of nanomaterials,the research on the use of nanomaterials for drug carriers has increased year by year.Also biosensors combined with advanced biomaterial manufacturing and biotechnology can build nanosensor recognition components for drug carrier[29]and drug delivery[30].The biosensor element consists of a micro switch and a control membrane.The drug delivery system is designed to release the drug when the sensor element is activated.At the same time,when combined with MEMS it can provide a broader prospect for the application of biosensor in the field of modern herbal medicine[31].

App lication and influence of biosensor as a new technology in action mechanism and effect of modern herbal medicine

The multicomponent action of traditional Chinese medicine has determ ined the complexity of its action mechanism,and there is still no effective way to elucidate the mechanism of modern herbal medicine.Based on the current research,the mechanism of traditional Chinese medicine is to regulate the function state balance of the cytokine network.The regulation of cytokines is achieved by activating its specific receptor.With the rapid development of biotechnology,especially the biochip technology and bio-sensing technology,newideas and methods are provided for the research of the mechanism of modern herbal medicine.Biochip technology,especially the development of high-throughput gene chips,can detect a large amount of information at the same time,which can help solve the problem of many targets of traditional Chinese medicine[32].However,there are still lim itations in the research on the mechanism.For example,the lack of effective analytical techniques or means,as well as the lag in gene expression and regulation research which make it difficult to explain the meaning of these genes at the same time[33].Biosensors use immobilized organisms or its content as a sensitive element to study the mechanism of traditional Chinese medicine,especially by affinity type biological sensing technology,which has played an increasingly important role.Lv Genfa et al.constructed a novel affinity biosensor for the screening and separation of antiendotoxin monomers in Paeoniae Radix[34].Xin Liu et al.constructed a kind of affinity biosensor to screen and isolate the active monomers and components of anti-sepsis[35].Xiuli Zhang et al.building an unmarked cell table analysis resonance waveguide grating(RWG)biosensor and successfully applied to identify(Paederia scandens)and(M illettia pachyloba)Drake effective component research[36].Biological sensing technology with its high specificity and affinity type can realize complex samples rapid detection and analysis of the active ingredients in Chinese medicine,compared with the traditional analysis method,it can greatly save the time and improve the detection sensitivity.

The pharmacodynam ics of traditional Chinese medicine is to study the biochemical,physiological and physiological effects and the relationship between dose and effect.The purpose of this study is to determine the efficacy of traditional Chinese medicine components in clinical prevention,diagnosis and treatment,determine the strength of each component;to clarify the target and correlation mechanism of each component;to find and use pharmacological effects for a wide range that outside of clinic[37].At present,the research on pharmacodynamics of traditional Chinese medicine has reached the molecular level of gene and protein and already made great progress.It is important to establish pharmacodynamics mathematical model of modern herbal medicine by using biosensor technology and comprehensive multidisciplinary association.It is of great significance to build comprehensive pharmacodynam ic evaluation system on the whole,cell and molecule,and to promote the modernization of Chinese medicine.

App lication of biosensors in quality evaluation of modern herbal medicine

In recent years,cultured traditional Chinese medicine(TCM)have gradually become the main resource of Chinese medicine industry due to the drastic reduction of wild herbal resources and the increasing demand for the stability and controllability of herbal medicine.The high pesticide residues,overproof heavy metals caused by improper manufacturing method and environmental pollution,and the toxic substances in the herbs all have impacts on the safety and effects of TCM.Therefore,it is crucial to monitor the quality of traditional Chinese medicine.Breeders and owners use high insecticidal pesticides frequently to prevent diseases and insect pests resulting in increasing pesticide residues,which affect the quality of traditional Chinese medicine seriously.

Residues of organophosphorus pesticide(OP)exist widely in TCM in China,and this class of toxic compounds inhibit the activity of acetylcholinesterase in humans,leading to respiratory and myocardial malfunctions and even death because of accumulation of the acetylcholine(ACh)neurotransm itter[38].Colorimetry[39],mass spectrometry(MS)[40],high-performance liquid chromatography(HPLC)[41]and thin layer chromatography[42-43]are some traditional methods employed to trace OP residues and monitor environmental pollutions.Biosensors have been widely applied in the detection of pesticide residues in recent years since their high efficiency and sensitivity.N.Chauhan et al[44]successfully constructed a biosensor with low detection lim it for determ ination of Malathion and chlorpyrifos by modifying gold electrode with immobilized acetylcholinesterase on ZnS.P.Raghu et al[45]prepared Silicasol–gel film/carbon paste electrode using encapsulation to determ ine the concentration of Methyl parathion and acephate.Our team fabricated an acetylcholinesterase biosensor by a layer-by-layer deposition technique for determ ining Trichlorfon[46].The study obtained a high activity enzyme by screening and prepared high performance immobilized enzyme electrode by a layer-by-layer deposition technique.A new technique of immobilization used for immobilizing more than two kinds of enzymes had been formed and biosensors with high sensitivity for choline,acetylcholine,residual organophosphorus pesticides were also constructed successfully[47].

Traditional Chinese medicine may be contam inated with lead in different degrees during the processing,storage and preparation.Soluble lead in water decoction and migrated lead under environment of gastric acid in human after swallowing TCM are related to the health of patients.In foreign countries,the trade standard is set up according to the total lead in traditional Chinese medicine,while a trade barrier emerges which restricting the export of Chinese medicine.Therefore,the analysis of valid lead and total amount of lead in TCM have always been the key point in quality control of Chinese medicine.Peptides and nucleotides have the capacity of selectively combining with Pb2+,which can be used for a series of biosensors to identify lead[48].Since the guanine residues and lead ion were able to form specific four dimer structures,Marta Jarczewska et al[49]modified the surface of gold electrode with guanine-enriched ssDNA probe and successfully constructed biosensor for detection of lead ion.The calculated lim it of detection was of 34.7 nmol/L.

The prospect of biosensor technology in the app lication of traditional Chinese medicine

Based on biosensor technology,our group has been conducting researches on traditional Chinese medicine for nearly 20 years.The detection of active components of various Chinese herbs was achieved.Our team constructs a set of research system applied to the mechanism of Chinese medicine,and proves the theory and results of TCM from the level of molecular biology.We have repeatedly undertaken the National Natural Fund Project on the new technology of traditional Chinese medicine.Two national invention patents have been obtained in the research of the mechanism of traditional Chinese medicine by our team.Therefore,the application of biosensor technology to traditional Chinese medicine is practical and practical,and has obvious advantages in many aspects.

With the rapid development of traditional Chinese medicine,more and more research achievements will be produced and human's daily life will be improved.Progress in one area also depends on the assistance of technical means.As an advanced method,bisensing technology,especially in the research of traditional Chinese medicine,is bound to make a great contribution to the development of traditional Chinese medicine.

Conclusion

In conclusion,the international profile and importance of traditional Chinese medicine is rising constantly with the development of people's cognition of TCM,and the study of TCM deserves immediate attention with significant resources.The modernization of Chinese medicine is an inevitable trend and in dire need of an efficient technology to boost the study of TCM.Biosensors have several advantages like high sensitivity,strong specificity,real-time and rapid detection thus facilitating various segments in study of herbal medicine,such as purification,identification of active compound,study of mechanism and quality control of TCM.Based on the practices and researches our laboratory made on biosensors,biosensors promise to be a powerful tool to study active compound and find therapeutic targets of TCM.With the deeper related theoretical discussion and wider use of technology method,biosensors will play to their strength and be applied widely in the research and modernization of Chinese medicine.