The Construction and Improvement of Novel Highly Sensitive Molecularly Imprinted Electrochemical Sensor

Author:Ma Ya

Supervisor:wang hai shui


Degree Year:2018





Molecularly imprinted polymer(MIP)is a class of new polymer materials with specific recognition capabilities,which has attracted widespread attention in recent years for the binding sites that match the target in the functional group and spatial structure,simple to prepare,good stability and can be used in the harsh environments such as acid,alkali,high temperature,and high pressure.MIP based electrochemical sensor also have the characteristics of good selectivity,high stability,low cost and long service life.At present,although there are many reports on molecularly imprinted electrochemical sensors,there esix some problems,such as the preparation methods are still not rich enough,the sensitivity needs to be improved,and when it comes to the template molecule,there are few studies focus on protein and other biological macromolecules.Therefore,a serious novel molecularly imprinted electrochemical sensors by introducing nanomaterials,nanocomposites and new detection methods for the highly sensitive detection of macromolecules protein were constructed.The main contents and results are as follows:(1)Human immunodeficiency virus p24(HIV-p24)antigen is a protein with great value in the prevention and control of AIDS.Based on this,a molecularly imprinted electrochemical sensor for the detection of HIV-p24 was prepared.First,multi-walled carbon nanotubes,chitosan,and glutaraldehyde were sequentially modified onto the surface of the electrode to not only increase the electron transfer rate between the electroactive material and the electrode but also capture a large number of target proteins efficiently,then the imprinted polymer was directly synthesized on the surface of the modified electrode through an in situ polymerization method,after the removal of template protein,a molecularly imprinted electrochemical sensor for HIV-p24 was obtained.The research on the identification mechanism and application of this sensor shows that,the introduction of multi-walled carbon nanotubes can significantly improves the sensitivity of the sensor,shortens the response time,as well as reduces the limit of detection(LOD).Under the optimized experimental conditions,the linear range of the sensor was 1.0×10-4 to 2 ng·m L-1 with the LOD of 0.083 pg·m L-1.Meanwhile,the MIP sensor also shows good selectivity,stability and reproducibility.For the detection of HIV-p24 in actual samples,the recovery rate is between 98.2%and 102.4%and the relative standard deviation is within 2.5%.(2)Consindering the excellent electrical properties,large specific surface area,and strong adsorption capacity of nanomaterials,graphene,multi-walled carbon nanotubes,and chitosan were modified to the surface of the electrode layer-by-layer.The synergy between them not only makes the conductive properties of the electrode improved greatly,but also provides more sites for the attachment of the template molecules to the electrode surface.In this work,we choose cardiac troponin c(cTn?)as template,under optimized experimental conditions,cTn?molecularly imprinted polymer membrane was directly synthesized on the electrode surface by in situ polymerization method.After removing the template molecule,a highly sensitive and selective molecularly imprinted electrochemical sensor for cTn?was obtained.The sensor provided a good response for cTn?in the concentration range of 0.005 to60 ng·mL-1 and a low limit of detection of 0.0008 ng·m L-1.When the sensor was applied for the determination of target in real samples,the results were in good agreement with the reference values,indicating a certain application prospect in the field of clinical analysis.(3)Prostate-specific antigen(PSA)is the best serum marker for detecting prostate cancer and breast cancer.Due to the advantages of simple operation and controlled thickness of film in the electropolymerization process,the sensor was prepared by using dopamine as an electropolymerization functional monomer and PSA as a template molecule,and then the molecularly imprinted membrane was formed on the surface of a graphene nanosheet-gold nanoparticle composite modified glassy carbon electrode by using cyclic voltammetry in a buffer solution.After the removal of template molecule,a PSA molecularly imprinted electrochemical sensor was obtained.The proposed sensor exhibited a linear response range from 1×10-3 to 100 ng·mL-1 for PSA with a limit of detection of 1.5×10-4 ng·m L-1.The sensor also exhibited convenience,low cost,rapidity,good specificity,acceptable stability and reproducibility.When it was used for the determination of PSA in human serum samples by addition recovery method,the recoveries ranged from 92.0%to 103.5%and RSD ranged from1.1%to 3.0%,indicating a certain guiding significance for PSA detection in clinical analysis.(4)When selecting the blot template,it was thought that a protein with a relatively stable configuration could achieve better imprinting results.However,for a class protein with variable conformations,such as gp120,is not considered because it is difficult to achieve accurate imprinting and effective identification.In this experiment,the conformation of gp120was stabilized by the introduction of a small molecule inhibitor NBD-556,then NBD-556@gp120 instead of gp120 was chosen as the template molecule,polymer film was prepared by electropolymerization of pyrrole onto carbon nano-fragments(CNF)and cerium oxide(CNF-Bi)composites-modified electrodes.After the template was eluted,a molecularly imprinted electrochemical sensor for highly sensitive detection of gp120 was prepared.Under the optimized experimental conditions,the sensor has a linear range of 0.002 to 200 ng·mL-1and a detection limit of 0.0003 ng·mL-1.It also shows good selectivity,reproducibility and stability.When the sensor was used in the actual serum samples,the spike recoveries ranged from 98.7%to 102%,with an RSD of less than 3.5%.Compared with the ELISA analysis method,the two have good agreement and the error is within an acceptable range at 95%confidence level.(5)Impedance-type molecularly imprinted electrochemical sensors are based on traditional frequency response analyzers(FRA)when testing for electrochemical impedance spectroscopy(EIS).They require the tested system in equilibrium state,so they cannot be used for the detection of dynamic process.On the other hand,excessively long test time may result in inaccurate test results or distorted spectra.In order to solve this problem,we introduce the dynamic impedance spectroscopy measurement technology into the MIP electrochemical sensor.With the home-made dynamic impedance spectroscopy instrument,the impedance type molecularly imprinted electrochemical sensor can be used to detect the dynamic process of the imprinting process,and through the testing of the dynamic characteristics,the early quantitative detection of macromolecular proteins in the non-equilibrium state can be achieved.Specific experiments was using bovine serum albumin(BSA)as an example,electropolymerization of pyrrole monomer and template molecule solution to the bare glassy carbon electrode surface,then remove the template molecule to obtain the imprinting sensor.The obtained sensor also shows high sensitivity,good reproducibility,good stability,and accuracy comparable to high-performance liquid chromatography(HPLC),and therefore has a good application prospect.