Study On Nanochannel Biosensor For The Detection Of Lead Ions In Traditional Chinese Medicine And SARS-COV-2

OBJECTIVE Nanochannel biosensor can achieve a label-free and highly sensitive detection of targets with its unique three-dimensional structure.By this new method,the development and application are worth exploring in the detection of the Traditional Chinese Medicine（TCM）products of quality control and SARS-COV-2 detection.Lead is one of the toxic heavy metals which is included TCM in the Chinese Pharmacopoeia（2020 edition）as the crucial index for the quality evaluation of TCM.The current methods of the lead ions detection have poor selectivity and low sensitivity,so it is of great significance to develop a new method with high sensitivity and specificity.At present,there is no mature treatment for the COVID-19,and the public health interventions are mainly to prevent and limit the spread of the virus,so the early detection of SARS-COV-2 has become the key to control the spread of the COVID-19.However,the current methods of SARS-COV-2detection have long sample turnover time and high requirements for operators and detection environment,so it is necessary to develop a new method.Above all,we have constructed functionalized-nanochannel biosensors to achieve label-free and highly sensitive detection of lead ions and SARS-COV-2.Moreover,the inhibition of TCM active ingredients for inhibited pseudovirus infection on target cells can be monitored by this biosensor.It provide new methods for the quality control of TCM,SASRS-COV 2 detection and theoretical and logistical support for the treatment of COVID-19.Method 1.In this chapter,alumina nanochannel is used as the substrate to connect the peptide probe which specifically bind with the lead ions.Thus,the peptide modified alumina nanochannel biosensor was constructed to detect the lead ions in TCM with a high sensitivity.Firstly,the porous anodized aluminum membrane（PAAM）was firstly washed with ethanol and ultra-pure water using an ultrasonic cleaner.Then,they were soaked in HCl solution,rinsed with ultra-pure water,and dried under nitrogen.After that,the prepared PAAMs were modified with3-aminopropyl trethoxysilane（APTES）and glutaraldehyde（GA）.Finally,the sensor was wholly constructed after the peptide modification and BSA block.Finally,the target lead ions and the peptide probe were specifically combined for the detection,and the method was applied to the detection of the lead ions in TCM.The I-V curves,X-ray photoelectron spectroscopy（XPS）,contact Angle（CA）measurements,field emission scanning electron microscopy（FE-SEM）and circular dichroism chromatography（CD）were used to validate the surface modification of the nanochannel and the process of binding lead ions.Various lead ions concentrations were introduced to interact with the peptide-modified nanochannels to study the sensitivity and linearity range of the biosensor.As³⁺,Cd²⁺,Co²⁺,Cr³⁺,Cu²⁺,Fe³⁺,Hg²⁺,Mg²⁺,Mn²⁺,Ni²⁺,Zn²⁺samples were used to study the selectivity of this biosensor.Four traditional Chinese medicines（Astragalus membranaceus,liquorice root,American ginseng,and Honeysuckle）were selected to detect lead ions.The peptide modified PAAM nanochannel sensor was used to detect these TCM samples.Atomic absorption spectrophotometry（AAS）was used to detect the lead ions simultaneously.Finally,the results were compared to evaluate the sensor’s performance in detecting lead ions.2.In this chapter,the The PET conical nanochannel is prepared as the substrate to connect the SARS-COV-2 S antibody which specifically bind with the SARS-COV-2.Thus,the SARS-COV-2 S antibody modified PET conical nanochannel biosensor was constructed to detect the SARS-COV-2 with a high sensitivity.Firstly,the chemical etching of the PET membrane was conducted according to the asymmetric track-etching method.Then the track-etched nanochannel film was immersed in an EDC/NHS mixture solution to activate the carboxyl groups.And finally,the SARS-COV-2 S antibody was covalently bound to the nanochannel surface,and then unreacted binding sites were blocked using BSA.Finally,the SARS-COV-2 and the antibody probe were specifically combined for the detection,and the method was applied to the detection of the SARS-COV-2.The I-V curves,XPS,CA measurements,FE-SEM and Laser confocal Microscopy（LSCM）were used to characterize each step of the modification and binding of SARS-COV-2.The affinity of SARS-COV-2 S protein and its antibody was investigated by surface plasma resonance method（SPR）.MERS-Co V S protein,MERS-Co V N protein,H1N1 antigen,and SARS N antigen were used to study the selectivity of this biosensor.Surface acoustic sensor（SAW）and flow cytometry were further used to verify SARS-COV-2 antibody-modified nanochannel sensor.Various concentrations of SARS-COV-2 S protein were introduced to interact with the SARS-COV-2 S antibody modified nanochannels to study the sensitivity and linearity range of the biosensor in 1×PBS.To meet the clinical needs,various concentrations of SARS-COV-2 S protein were also introduced to interact with the SARS-COV-2 S antibody modified nanochannels to study the sensitivity and linearity range of the biosensor in throat swabs.The ability of this biosensor to distinguish between SARS-COV-2 and normal samples was also investigated.3.Based on the above chapter,the constructed PET conical nanochannel biosensor was used to monitor the active components of TCM to inhibit COVID-19.The 293T cells with high ACE2 expression were first constructed,and the ACE2 expression was tested by real-time PCR and WB to verify transfection results.Auto Dock Tools 1.5.6 was used for molecular docking analysis,and Py Mol software was used to visualize the binding interaction between the active ingredients of TCM（Alolide 1,Salvianolic acid B,baicalein,berberine,and quercetin）and SARS-COV-2S protein in a 3D model.The CCK-8 was used to screen the administered concentrations of five TCM active ingredients.Salvianolic acid B and baicalein were incubated with 293T cells for 6 h,and the same concentration of SARS-COV-2 pseudovirus was mixed at room temperature for 48h.Then,the fluorescence microscopy was used to investigate the infection of target cells.The sensor’s I-V curve variations were evaluated to determine if the sensor could monitor the inhibition of TCM active ingredients inhibited SARS-COV-2 pseudovirus infection.Result 1.The I-V curves,XPS,CA measurements,FE-SEM and CD results were suggested that the successful modification of the peptide,and the lead ions are also successfully captured.The I-V curves increased from 0.01-0.16μM and 10-100μM with the concentration of lead ions and showed a good linear correlation,with|I-I₀/I₀|=9.1942x+0.093（R~2=0.9950）and|I-I₀/I₀|=0.0513x+1.4006（R~2=0.9918）,and the detection limit was 0.005 M.This peptide-modified nanochannel sensor showed a good specificity for lead ions in different kinds of metal ions(As²⁺,Cd³⁺,Co²⁺,Cr²⁺,Cu²⁺,Fe³⁺,Hg²⁺,Mg²⁺,Mn²⁺,Ni²⁺,Zn²⁺).Moreover,this method was selective enough to be applied for detecting complex TCM samples.Compared with the AAS method,the recovery rate of the peptide-modified nanochannel sensor was 87.7%-116.8%.2.The I-V curves,XPS,CA measurements,FE-SEM and LSCM were suggested that the successful modification of the SARS-COV-2 S antibody,and the SARS-COV-2 are also successfully captured.The SPR results proved a good affinity between SARS-COV-2 S protein and SARS-COV-2 S protein,the value of the KD was 1.09 n M.The signal response of the MERS-Co V S,MERS-Co V N,H1N1 and SARS N proteins are lower than the SARS-COV-2 S protein,and the SAW and flow cytometry results found that the SARS-COV-2 S antibody had a higher response to the SARS-COV-2 S protein than the other antibodies.The above results showed good selectivity for SARS-COV-2 of this biosensor.1×PBS,there is a linear correlation between the SARS-COV-2S protein concentration logarithm and the current change value（1 fg/ml to 1 ng/m L）.The LOD was 0.67 fg/m L.In the throat swab samples,the ion current similarly increased with the concentration of SARS-COV-2 S protein,with the response range ranging from 10 fg/ml to 1 ng/ml.Moreover,this biosensor is capable of discriminating pseudoviruses and normal samples.3.We produced 293t cells with high expression of ACE2 by transfecting them with plasmids.The transfection results were evaluated by WB and RT-PCR,which confirmed that the transfection was effective.Molecular docking results found that the binding energy of 5 TCM active ingredients and SARS-COV-2 S protein was less than-6 kcal/mol.Salvianolic acid B and baicalein have been selected as the top two in the binding energy score,then tested with the fluorescence situation and our sensor,respectively.Compared with the blank group,both of salvianolic acid B and baicalein showed lower fluorescence intensity and higher rate change of current alterations.n conclusion of these data,our sensor can be used to monitor the inhibition of TCM active ingredients for inhibited pseudovirus infection.Conclusion 1.A peptide-modified nanochannel sensor was designed with high sensitivity and label-free to detect lead ions and the detection was selective enough to be applied to complex TCM samples.2.A track-etching conical nanochannels immunosensor was conctructed to detect SARS-COV-2 with high sensitivity and selectivity.Moreover,this sensor can be used for the detection of swab samples.3.The inhibition can be monitored by PET conical nanochannel sensor,providing new ideas for further exploring the development of TCM for the COVID-19 treatment.