Construction And Performance Of Highly Sensitive Pb²⁺ Bio-machine Based On GR-5 Dnazyme And Isothermal Amplification

With the progress of industrialization,toxic metal contaminants,especially lead（Ⅱ）ion(Pb²⁺),were widely distributed in the ecological environment,which seriously threatens environmental safety and public health.Long-term exposure to Pb²⁺will cause irreparable damage to the nervous system,growth and development system,cardiovascular system,and immune system of the human body,and then lead to diseases such as body dysfunction and neurodevelopment,which is particularly harmful to children.Therefore,people have paid great attention to the monitoring and treatment of lead metal pollution.However,traditional large-scale precision instruments are difficult to meet the urgent needs for fast and sensitive on-site real-time detection of lead pollution.Thus,it is of great significance to develop highly sensitive and convenient on-site lead ion detection methods and technologies.Deoxyribonuclease（DNAzymes）is an in vitro enrichment-screening of catalytically active oligo DNA.It has the advantages of high chemical stability,strong programmability,easy synthesis,easy modification,and low cost,etc.,which is one of the ideal components for constructing a highly sensitive biosensor platform.It is an important element for building highly sensitive biosensor platforms.To date,colorimetric,fluorescent,and electrochemical biosensor probes based on DNAzymes have attracted a lot of research interest and have been widely studied and reported.However,the sensitivity and portability of many detection platforms cannot meet the requirements of field detection.The isothermal nucleic acid amplification strategy eliminates the tedious operation processes of PCR amplification,such as temperature programming and temperature control,and does not require accurate temperature control instruments.So,many types of isothermal amplification technology have been widely used to increase the signal intensity of biosensors,such as hybridization strand reaction（HCR）,isothermal strand displacement amplification（ISDA）,rolling circle amplification（RCA）,nicking enzyme signal amplification（NESA）,and DNAzyme assisted feedback amplification.Among them,the design and application of ISDA in biosensors are the most common,because of its highly efficient amplification performance,easy operation,and high specificity.In this paper,using DNAzyme as recognition element and isothermal strand displacement amplification as signal amplification strategy,two highly sensitive,fast,simple,and flexible lead（II）ion detection platforms are constructed and their detection performance is studied and discussed.The main work includes the following:1.Based on extended GR-5 DNAzyme autonomous isothermal amplification machine（EGD-AICM）for highly sensitive Pb²⁺colorimetric detection.In this chapter,we proposed a novel extended GR-5 DNAzyme-based autonomous isothermal cascade machine（EGD-AICM）for one-tube high-sensitivity analysis of trace Pb²⁺.We integrate GR-5 DNAzyme,the specific sequence for nicking by Nb.Bpu10I,and an antisense EAD2 G-rich DNA sequence into one molecule（extended GR-5 DNAzyme,GR-5E）.3′-end of the GR-5 substrate is extended（extended GR-5 substrate,GR-5S）with three bases（AAA）that mismatch with GR-5E,which endows an excellent specificity of EGD-AICM toward target Pb²⁺.EGD-AICM combines the recognition ability of GR-5DNAzyme with signal amplification/transduction capabilities of ISDA and horseradish peroxidase（HRP）-mimicking DNAzyme.Pb²⁺activates EGD-AICM to implement autonomously replication-scission-displacement cycles and further generates numerous EAD2.Therefore,EGD-AICM enables label-free and one-tube determination of Pb²⁺with a short assay time（～50 min）,excellent selectivity,and high sensitivity（LOD=35.25p M）.In addition,the proposed EGD-AICM has been employed to measure Pb²⁺in real water and shrimp samples with satisfactory accuracy and precision.Such a straightforward and economical EGD-AICM platform demonstrates great potential for routinely screening metal ions in environmental monitoring,food safety assessment,and public safety fields.2.Construction of a feedback cascade amplification machine（FICAM）based on extended GR-5 DNAzyme and molecule beacon（MB）for Pb²⁺high sensitivity fluorescence detectionIn this chapter,a feedback cascade amplification system was constructed based on self-designed GR-5 DNAzyme and molecule beacon.First,the MB,GR-5 E1,GR-5 S1was designed.MB is an oligonucleotide composed of 41 bases,a 5’-end modified fluorescent group（FAM）,and a 3’-end modified quenching group（DABCYL）.The neck of MB is composed of It consists of 11 pairs of bases,the loop consists of 19 bases with Nb.Bpu10I recognition sequence.Then,according to the target sequence of MB molecule,a DNAzyme（GR-5E1）capable of recognizing Pb²⁺and its corresponding substrate（GR-5S1）are designed.When lead ions exist,GR-5E1 recognizes the lead ions and cuts the adenine nucleotide（r A）site of GR-5S1,so that GR-5S1 is divided into two parts,and the5’end part is hybridized with GR-5E1 as an isothermal extension primer,making it isothermal strand replacement process initiates and releases a large number of Target DNA oligonucleotides.Then,the generated DNA strand hybridizes with hairpin DNA（named:target-MB）and releases a fluorescent signal.At this time,the 3’end fragment of GR-5S1 and target-MB hybridize with each other to serve as primers for isothermal cascade amplification,and then MB is used as a template to perform isothermal amplification again to generate target single-stranded DNA again.By designing such a cascade amplification process through a strand displacement amplification technology,the detection sensitivity and the signal-to-noise ratio in the detection process can be significantly improved.Under optimized conditions,the linear ranges of detection of Pb²⁺are 1 p M-1n M and 1 n M-100 n M,and theoretical detection limits are 0.118 p M and 0.055n M.This method shows good recovery（98.90%-108.20%）and low detection error in real sample detection.The biosensor platform has great potential for the detection of trace Pb²⁺in the environment and food.