Construction Of Novel Aptamer-piezoelectric Sensor And Its Application For Detection Of Mycobacterium Tuberculosis

Tuberculosis（TB）is a chronic serious infectious disease with Mycobacterium tuberculosis infection that seriously endangers human health and is a public health and social problem of global concern.The TB pandemic occurred during the industrial revolution in Europe in the 18th century,and is still the greatest public health challenge in the world.At present,there are 1.3 million new cases in the world every year,and one causes of death every15 seconds.The epidemic situation of tuberculosis in our country is also very serious.There are 130 thousand new TB cases in the country every year,and the number of TB cases in China ranks the third in the world.What’s more,the problem of drug-resistant tuberculosis has become more and more serious in recent years.The number of MDR-TB in China is the second in the world,with about 120 thousand new cases every year.Early diagnosis of TB is an effective measure to prevent and cure TB,and is one of the key steps to control the transmission of TB.The aptamer is a single-stranded DNA or RNA oligonucleotide screened by SELEX technology in vitro.It has high specificity and affinity recognition and binding ability to the target,and is easy to be chemically synthesized,reproducible,modified,and stable at room temperature.It is also reusable at room temperature,even after denaturation and renaturation.These unique advantages can overcome the restrictions of antibodies detection methods.As molecular devices,affinity separation materials,and therapeutic agents,aptamer has been widely applied in biosensors.In view of this,the Mycobacterium tuberculosis H37Rv aptamer was screened and modified as a recognition probe on the electrode of a multi-channel piezoelectric quartz crystal（MSPQC）sensor to achieve rapid detection of non-culture of Mycobacterium tuberculosis H37Rv.The sensitivity of the detection is further improved by means of single-walled carbon nanotubes,gold nanoparticle and DNA-nanoparticle.MSPQC sensor can sensitively detect changes in electrical parameters and is widely used in the analysis of microorganisms because of its advantages of fastness,sensitivity,simple operation and low cost.A series of sensors based on MSPQC system were designed to detect Mycobacterium tuberculosis.Although the detection time has shorten compared with traditional method,the demand of rapid detection is still unmet owing to the lengthy culture period.On the basis of the previous research of our group,this project is devoted to the development of a new MSPQC sensor that is sensitive,fast,accurate,inexpensive and easy to popularize.The rapid detection of Mycobacterium tuberculosis will be realized by introducing aptamers as detection probe without culture.In view of this,some research work has been carried out:（1）The screening strategy of H37Rv aptamers was established by cell-SELEX technology.Using H37Rv as a screening target,a ssDNA library with a total length of 78 nt bases and 35 nt random sequences in the middle was designed in vitro.The library capacity was 4³⁵ to ensure that aptamer library is large enough to obtain abundant sub-library.The double-stranded DNA was prepared by symmetric PCR,and then used as a template to carry out asymmetric PCR,and the product was purified by electrophoresis,thereby ensuring the quality and quantity of the sub-library,and purifying and separating with an electrophoresis gel to ensure the effect.From the fourth round,Mycobacterium smegmatis,Staphylococcus aureus,Salmonella typhimurium and Escherichia coli were used as targets for reverse screening,which improved the binding rate between sub-libraries and target bacteria.At the same time,in order to improve the screening effect,the input of sub-libraries and Mycobacterium tuberculosis was reduced by turns,and the test conditions of PCR were optimized.The degree of enrichment of aptamers was monitored by Fluorescent spectrophotometer assay and Flow cytometer assay.（2）Cloning,sequencing and identification of the aptamer of Mycobacterium tuberculosis.The library of the 14th round screening elution enrichment was cloned and sequenced.Forty clones were obtained through blue and white spot test,and 39 effective aptamer sequences were obtained.The primary structure sequence and secondary structure of the library were compared and analyzed,and secondary structure of the library was simulated.Four sequences with the most stable structure were selected for affinity determination by fluorescence analysis.Among them,the aptamer Apt1 had the highest affinity,and the Kd value was 37±4nmol/L.The FITC-labeled aptamer Apt1 was used for quantitative and specific investigation of Mycobacterium tuberculosis.The experiment results exhibited that the aptamer was highly specific to Mycobacterium tuberculosis and did not cross-bind to Mycobacterium smegmatis with highly homologous genes and similar cell structure.The quantitative assay of H37Rv exhibited that there was a linear relationship in the range of 10²¹0⁷cfu/mL.（3）Based on the specific recognition of Mycobacterium tuberculosis by its aptamer,the probe was produced by immobilizing thiol-modified aptamer on an Au interdigital electrode（Au-IDE）of MSPQC through Au-S bonding,and then single walled carbon nanotubes（SWCNTs）were bonded on the aptamer byπ-πstacking.SWCNTs were used as a signal indicator because of their considerable difference in conductivity compared with H37Rv.When H37Rv is present,it replaces the SWCNTs because it binds to the aptamer much more strongly than SWCNTs do.The replacement of SWCNTs by H37Rv resulted in a large change in the electrical properties,and this change was detected by the MSPQC.The proposed sensor is highly selective and can distinguish H37Rv from Mycobacterium smegmatis and Bacillus Calmette-Guerin vaccine.The detection time was 70 min and the detection limit was 100cfu/mL.Compared with conventional methods,this new SWCNTs/aptamer/Au-IDE MSPQC H37Rv sensor was specific,rapid,and sensitive,and it holds great potential for the early detection of H37Rv in clinical diagnosis.（4）DNA-AuNPs/MSPQC sensor was constructed based on the high specificity and affinity of Mycobacterium tuberculosis aptamer Apt1 to H37Rv.AuNPs were modified on the interdigital gold electrode surface of MSPQC by capture probe.When H37Rv was present,the detection probe Apt1 specifically recognized that H37Rv formed Apt1-H37Rv complex,and connected Apt1-H37Rv complex to the electrode surface by complementary hybridization of the aptamer Apt1 and capture probe,which resulted in the impediment of charge transfer on the electrode surface.The frequency response of MSPQC sensor is caused by the change of electrical parameters.The sensor can specifically recognize H37Rv in the coexistence of Bacillus Calmette-Guerin vaccine and Mycobacterium smegmatis.It can detect H37Rv without culture.The detection time is 65 minutes and the detection limit is 100 cfu/mL.Compared with traditional methods,this method is fast,sensitive and specific,and has important application prospects in early clinical diagnosis of H37Rv.（5）To construct an AuNPs-DNA mediated H37Rv aptamer/AuNPs-DNA/MSPQC sensor using H37Rv aptamer as recognition probe.Three gold nano-modified oligonucleotides were designed.These oligonucleotides contained 12,12,and 13 bases that hybridized with the37-nt H37Rv aptamer.H37Rv aptamer was immobilized on the gold electrode surface by Au-S bonds.A conductive-layer was then formed by sequential hybridization of the aptamer with the three designed AuNPs-DNAs.When H37Rv was present,it specifically bound to the aptamer,resulting in the detachment of AuNPs-DNA from the electrode.The conductive layer was thereby replaced by a non-conductive complex of aptamer and bacteria.These changes were monitored by the MSPQC system.The proposed sensor is rapid,specific and sensitive,the detection time was 2 h.The limit of detection limit was 100 cfu/mL.This sensor would be of great benefit for the early clinical diagnosis of tuberculosis.