Applications Of A Novel Field Effect Transistor Biosensor In Monitoring Heart Failure-related Markers And Calcium Ion Release From Cardiomyocytes Regulated By Traditional Chinese Medicine

With the development of China’s population aging,the incidence of cardiovascular and cerebrovascular diseases increases year by year,and most cardiovascular and cerebrovascular diseases will eventually lead to heart failure(HF).HF is a pathological process caused by low myocardial contraction-relaxation capacity,resulting in a relatively reduced cardiac output.In this process,the increase in intraventricular pressure and ventricular wall tension in patients with HF leads to the release of brain natriuretic peptide(BNP).The BNP content in the blood increases from normal levels 6 pM to severity HF 600 pM.In addition,with cardiac cycle changes,the most important second messenger calcium ion(Ca2+)in cardiomyocytescan regulate intracellular Ca2+ concentration by Ca2+ absorption,storage and release through the sarcoplasmic reticulum,thereby regulating the excitatory-contraction of the process of myocardial contraction.When any one of three links gets obstacles,it will affect the excitatory-contraction process,resulting in decreased myocardial contractilityand causing HF.Thus,highly sensitive detection of BNP in whole blood and real-time monitoring of intracellular Ca2+ concentration are beneficial to the early diagnosis and prevention of HF.Studies have shown that ginseng total saponins(GS)can effectivelyimprove hemodynamics,inhibition of ventricular remodeling,thus protecting the heart function.Berberine(Ber)has a good protective effect on the occurrence of HF and the energy metabolism of myocardium.The two active ingredients of traditional Chinese medicine have an impact onthe plasma BNP levels and myocardial Ca2+ concentration.Since "ginseng and berberine" are classic pairs of the drugs for Chinese medicine,many studies are committed to explore the function of the two traditional Chinese medicine on prevention and cure of HF.Therefore,the study of total ginsenoside combined with berberine can control the release of Ca2+ in cardiomyocytes and help modern Chinese medicine to prevent and cure HF.In recent years,with the vigorous development of nanoscience and interfacial science,the field effect transistor(FET)biosensor based on nanomaterials has attracted great attention.Beacuse its research involves biology,information,physics,chemistry and it has a revolutionary impact on many important areaslike clinical testing,genetic analysis,environmental testing,biological anti-terrorism and national security,FET biosensor becomes the hot field of the international research.Nano-materials have the advantages of large specific surface area,strong adsorption capacity,high catalytic efficiency and high surface active sites.In particular,graphene exhibits more excellent physical and chemical properties than other nanomaterials,in terms of excellent conductivity and electrocatalysis performance,large surface area,good biocompatibility and mechanical properties,etc.These excellent properties make graphene ideal for the preparation of graphene-based FET biosensors.Therefore,a new biosensor based on graphite-based FET can provide a new method for the detection of HF-related markers.In this paper,a new type of field-effect transistor biosensor based on platinum nanoparticles(PtNPs)modified reduced graphene(RGO)is proposed for highly sensitive and highle specific detection of heartfailure-related biomarker,BNP.Moreover,another FET biosensor based on reduced graphene/Titanium dioxide(RGO/TiO2)for real time monitoring Ca2+released from cell is also reported.This will help understand the change of Ca2+ concentration inside and outside of cells.Meanwhile,Ca2+ release in cardiomyocytes regulated by GS and Ber is monitored by the functionalized FET biosensor,which provides theoretical and technical support for the treatment of HF.Part 1: Detection of heart failure-related biomarker in whole blood with graphene field effect transistor biosensorFirst,a 50 nm thick gold electrode was deposited on a silicon wafer by photolithography and electron beam evaporation coating to prepare a transistor chip.GO was synthesized by the traditional Hummer preparation method.The synthesized GO was reduced under the action of hydrazine hydrate to produce RGO.In the sensing channel,RGO was deposited as a conductive channel by dropping method,and the p-type RGO FET biosensor was prepared by ultrasonic and annealing process.The mechanical properties of RGO in the sensing area were given by optical electron microscopy.The electrical properties of the device were measured by using the semiconductor analyzer.The transfer characteristic curve of the device was measured.Then,Pt NPs were deposited by UV irradiation in the RGO FET biosensor region.BNP antibody,as a probe molecule,was immobilized on the Pt NPs surface through Pt-S bond.The PtNPs/rGO FET biosensor could rapidly respond to BNP with high sensitivity and high specificity.Because BNP molecule is charged,BNP binding on the sensor surface via antigen-antibody reaction will lead to carrier concentration changes on graphene surface,thereby inducing the shift of the Dirac point in the transfer characteristic curve.Current signal decreased with the increased BNP concentration.It wasfound that the PtNPs/rGO FET sensors could specifically detect BNP and achieve a detection limit as low as 100 fM.The logarithmic value of BNP concentration defined as lgC showed a linear response within the range of 100fM-1 n M.The linear relationship was represented by ?I(nA)=-10.43lgCBNP(M)-140.40 and correlation coefficient was 0.9917.In general,the greater the BNP level in the blood,the more severe the heart failure condition.So the result reveals that the PtNPs/rGO FET biosensor could achieve high sensitivity,which is capable of detecting BNP in human whole bloodat the early stage of HF.Then,the custom-made microfilter was employed to remove blood cells for clinical blood samples.After blood solutions including 50,100,200 nM BNP,respectively,were filtered and applied to the PtNPs/rGO FET biosensors,it was clearly observed that response increased along with the increase of BNP concentrations in blood,clearly demonstrating the Pt NPs/rGO FET’s ability to detect the biomarkers in human blood sample.Part 2: Real-time monitoring of Ca2+ release from cell based on RGO/TiO2/RGO functionalized field effect transistor biosensorAt first,the TiO2 nanopartciles functionalized with APTMS were coated with thin layers of GO through the electrostatic interaction between the positively charged TiO2 nanoparticles and the negatively charged GO,followed by reduction with hydrazine to form RGO/TiO2 composites Meanwhile,the FET chip prepared by a traditional microfabrication process was made into G-FET by drop-casting RGO and the synthesized RGO/TiO2,to the chip surface between the source and drain electrodes as conductive channels.After these processes,the calcium ion probe molecule Fluo 4-AM was immobilized on the interface of the sensor by π-π stacking.After Ca2+ was bound to Fluo 4-AM,the conductivity of RGO/TiO2/RGO FET biosensor changed.By such,the developed biosensor could be used for real-time monitoring of Ca2+ released from human umbilical vein endothelial cells(HUVECs).Finally,the regeneration of the chip was realized by irradiating with ultraviolet ray,through which all the substances on the graphene surface were removed and the chip can be regenerated for the next run of detection.To determine the sensitivity,the Fluo 4-AM functionalized FET biosensor was used to monitor Ca2+ in PBS and FBS,respectively.In PBS solution,the logarithmic value of Ca2+ concentration defined as lgC showed a linear response within the range of 100 pM~1 mM.The linear relationship was represented by ΔI(nA)=36.97lgC(M)+363.08.The detection limit of Ca2+was determined to be 100 pM.In FBS solution,the logarithmic value of Ca2+concentration defined as lgC showed a linear response within the range of 1nM~1 mM.The linear relationship was represented by ?I(n A)=32.49lgC(M)+332.43.The detection limit of Ca2+ was determined to be 1 nM.Finally,the Fluo 4-AM functionalized RGO/TiO2/RGO FET biosensors was used for monitoring of the release of Ca2+ from human umbilical vein endothelial cells(HUVECs).It was found that the current signal decreased when the cell was stimulated by high potassium solution,making Ca2+released from the cell.As known,TiO2 is wildly used as photocatalysis-induced cleaning material because it is capable of decomposing the organic molecules by active oxygen radicals generated under ultraviolet irradiation without altering the surface morphology and structure.It was found that the Fluo 4-AM functionalized FET biosensor had the ability of reusability via photo catalysis,providing a new renewable method for monitoring of Ca2+release from cell.Part 3: Monitoring of Ca2+ release from cardiomyocytes regulated by total ginsenoside and berberineThe active ingredients GS and Ber of the two Chinese medicine ginseng and berberine were used to culture with cardiomyocytes and the Fluo 4-AM functionalized RGO FET biosensor was then employed to monitor the releaseof Ca2+ from the cell.The cells were cultured with Ber,GS,GS+Ber and the serum-free medium,respectively.The FET biosensor was used to monitor the release of Ca2+ from cardiomyocytes in a real time manner.The results showed that the concentrations of Ca2+ in cardiomyocytes cultured with Ber,GS and GS+Ber decreased,respectively,because the three groups of drugs had inhibitory effect on Ca2+ concentration in cardiomyocytes,making the intracellular Ca2+ concentration decreased.The changes of Ca2+ concentration were measured by the percentage of electrical signal decline.Ber,GS and GS+Ber decreased the intracellular Ca2+ concentration by 22.4%,27.1% and41.5% respectively.Ber can reduce Ca2+ concentration in myocardial cardiomyocytes and play a role in improving diastolic function.While,GS can protect mitochondrial calcium pump activity inmyocardial cells,protect membrane system integrity,reduce intracellular free Ca2+ concentration,and prevent internal calcium in myocardial cells overloaded.The synergistic effect of Ber and GS can play a role in improving calcium overload in myocardial cells.The results showed that the Fluo 4-AM functionalized RGO FET biosensor could be applied in real-time monitoring Ca2+ release from cardiomyocytes,providing a new method for evaluating the efficacy of traditional Chinese medicine in HF.