Detection Of Drug Efficacy And Side-effects With Cardiomyocytes-based Biosensors And Application Study

The problem of drug safety is a major challenge at present.It is of great relevance to human health and safety,but adverse events caused by drugs occur every year.Among them,cardiac side-effects caused by drugs are one of the major factors that terminate new drug research and development and withdraw listed drugs from the market,which pose serious threats on public health and economic development.For the preclinical drug cardiac safety studies,animal models largely rely on manual operation because of the complex drug evaluation processes,which limits the throughput and the experimental efficiency is subsequently insufficient.Therefore,many high throughputs and functional cell-level drug evaluations have been urgent in recent years.In vitro tests,especially using myocardial cells as the analysis object,can not only give accurate judgment on the cardiac side-effects of drugs in the early research and development stage,thus providing a reference basis for the formulation and selection of subsequent trials,but also provide safe and trustworthy track for the drugs that have been listed already.A comprehensive evaluation is also greatly significant to reduce the cost of drug development and shorten the cycle,as well as reduce the risk of clinical subjects,and finally ensure the credibility of drugs.Laboratory-based cell viability assays usually use labeled methods for analysis,such as fluorescent dyes(fluorescein,ionic dyes),reducing compounds(CCK-8,MTT reagents),and etc.,which reflect cell behavior only in single time points.Likewise,many well-developed engineering technologies such as voltage clamps,patch clamps,and microelectrode arrays can only extract a single parameter.Therefore,novel methods to achieve high-throughput,continuous and multi-parameter evaluation of drug safety are urgently needed.This thesis is based on cell-based biosensor technology and uses HL-1 cardiomyocytes as sensitive components.A new multi-channel biosensor is studied.A dual-function detection system integrating cell electrical impedance and electrophysiological excitability,and corresponding analysis signals processing algorithm are designed under the new in vitro heart model.Finally,this work selects two typical clinical drugs for test,achieving the detection and evaluation of drug efficacy and cardiac side-effects simultaneously.The main research contents and innovative work completed in this thesis are listed as follows:1.A new type of multi-channel and dual-functional cardiomyocyte-based biosensor was designed and fabricated,which is capable of the composite sensing of both cell electrical impedance and extracellular field potential signal.In this work,a new multi-channel myocardial cell-based biosensor was designed,with each sensing unit including two pairs of fine-pitch interdigitated electrodes(IDEs)and two sets of planar microelectrodes.For each of them,IDEs are used for cell electrical impedance sensing,which is able to track the change induced by like the number of cells attached to the electrode surface,morphology growth,and intercellular connection and so on.The planar microelectrode is hereby used for cell extracellular field potential signal sensing,which detects,especially among the excitable cells,the electrophysiological activities and changes caused by the cross-membrane transportation of ions at the surface.Combined with lift-off process technology,the proposed new multi-channel cardiomyocytes-based biosensor can achieve composite sensing of cell electrical impedance and extracellular field potential signals within the same unit simultaneously.2.A new detection and control system integrating cell viability and electrophysiological excitability was designed,including the corresponding multi-parameter signal analysis software of cell electrical impedance and extracellular field potential respectively.In order to meet the needs of the designed multi-channel cardiomyocytes-based biosensor capable of combined sensing of electrical impedance and extracellular field potential signals,this work set up a matched dual-function hardware detection and control circuit system,realizing the simultaneous detection and track of cell viability and electrophysiological excitability.The coefficient of variation(CV)of the cell electrical impedance detection module is 0.5%,and the long-term drift over a 24-hour period is less than 0.1 CI.The peak-to-peak noise of the extracellular field potential detection module is 10 μV.On this basis,this paper also developed a multi-parameter signal analysis algorithm supporting to achieve the cell viability tracking,as well as the extraction and analysis of periodic electrophysiological signal time-dependent features.3.A new high temporal range analysis model of cell viability and electrophysiological excitability combined with the new HL-1 cardiomyocytes was proposed,and the feasibility of the model was verified in the evaluation of clinical drug efficacy and cardiac side-effects.HL-1 cells are currently the only cardiomyocytes that can continuously divide and maintain the phenotypic characteristics of mature cardiomyocytes,which is introduced as the sensitive element in this work.The implantation density of the HL-1 cardiomyocytes on the multi-channel biosensor was optimized,and a high temporal range analysis model based on the principle of long-term cell viability and short-term electrophysiological excitability changes was proposed correspondingly.Parameters including the excitation frequency of the electrical impedance signal monitoring and periodic electrophysiological characteristics over multiple time periods were discussed in depth.Moreover,with the comparison of traditional bioassay,CCK-8 and live/dead methods,two clinical drugs,the cardiovascular drug Nifedipine and the chemotherapy drug Vinblastine were applied to verify the performance of the proposed model.Finally,the drug efficacy and cardiac side-effects evaluation were completed in a multi-parameters way.