Amplification-Free And Highly Sensitive Detection Of Viral Nucleic Acids Based On Graphene Field-Effect Transistor Biosensor

The rapid development of the field of laboratory medicine cannot be achieved without the innovation and breakthrough of new technologies and methods such as nano-biosensors.Among the variety of biosensors currently available,field-effect transistor（FET）biosensors,especially in combination with advanced functional nanomaterials,have been considered as ideal candidates for next-generation medical diagnostic platform,which allow the instant ultrasensitive detection while avoiding the dependence on various labelings.In recent years,the front-end development and clinical application of field-effect transistor（FET）biosensors continue to bring new ways and new ideas for the detection of many biomarkers and disease diagnosis,with great development potential and application prospects.FET is a three-terminal device containing a source（S）,a drain（D）,and a gate（G）.Its main operating principle is to regulate the conductance between the source and drain by applying a gate voltage to the gate.In detection,the FET induces an immediate electrical response by directly sensing the charge carried by the target analyte itself,thus enabling label-free detection and dynamic real-time analysis.In addition,FETs have the advantages of high sensitivity,small size,low power consumption,low noise,and easy integration,and are considered ideal candidates for next-generation medical diagnostic platforms.Graphene is a two-dimensional carbon-based nanomaterial in a honeycomb structure with high carrier mobility,large body surface ratio,strong electrical conductivity,bipolar field effects,and good biocompatibility.FETs with graphene as the conductive material can play a unique advantage in the recognition and detection of biomolecules and are one of the most widely used biosensors.Viral infectious diseases have been common and prevalent,and are one of the major factors leading to death in critically ill patients.In particular,the emergence and spread of severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）has posed a major threat to human life and health in recent years,and the importance of virus detection has once again aroused people’s attention.Nucleic acid testing,as it is known to the general public,has become the first port of call for confirming viral infections,and is used in medical decision making throughout the disease process.However,traditional tests such as RT-PCR are complicated and time-consuming,and are not suitable for rapid screening and bedside detection.Therefore,there is an urgent need to develop more rapid,effective,low-cost,and user-friendly detection strategies for timely and accurate diagnosis of COVID-19.Based on this,this dissertation establishes two novel virus detection platforms based on graphene field-effect transistor（G-FET）biosensors for amplification-free and highly sensitive detection of SARS-CoV-2,with the aim of bringing new approaches to the establishment and application of virus detection methodologies.This dissertation consists of the following two main parts:Part Ⅰ: PMO-functionalized graphene field-effect transistor biosensor for rapid,amplification-free detection of SARS-CoV-2In this part,we develop a rapid and unamplified nanosensing platform for detection of SARS-CoV-2 RNA in human throat swab specimens.A gold nanoparticle（AuNP）-decorated graphene field-effect transistor（G-FET）sensor was fabricated,after which complementary phosphorodiamidate morpholino oligos（PMO）probe was immobilized on the AuNP surface.This sensor allowed for highly sensitive testing of SARS-CoV-2 RdRp as PMO does not have charges,leading to low background signal.Taking advantage of the high surface-to-volume ratio of AuNP,and high stability,efficacy of PMO probe,trace amounts of SARS-CoV-2 RdRp gene can trigger a measurable shift of the Dirac point of the G-FET sensor upon hybridization of PMO with RdRp gene.Not only did the method present a low limit of detection in PBS,throat swab and serum,but also it achieved a rapid response to COVID-19patients’ samples within 2 min.The developed nanosensor was capable of analyzing RNA extracts from 30 real clinical samples.The results show that the sensor could differentiate the healthy people from infected people,which are in high agreement with RT-PCR results（Kappa index=0.92）.Furthermore,a well-defined distinction between SARS-CoV-2 RdRp and SARS-CoV RdRp was also made.Most critical of all,the tedious,laborious process of RT-PCR amplification is circumvented,performing a high-speed direct detection without cumbersome manipulations.Therefore,we believe that this work provides a satisfactory,attractive option for COVID-19 diagnosis.Part Ⅱ: Dual CRISPR/Cas13a-mediated graphene field-effect transistor biosensor for highly sensitive,amplification-free detection of SARS-CoV-2 RNACRISPR/Cas13 system,an innovative modality for RNA detection,has been advanced to a promising option for SARS-CoV-2 diagnosis.However,the long reaction time and the dependence on pre-amplification make it still far from an ideal diagnostic tool.CRISPR technology has been cross-fertilized with new technologies and methods such as nano-biosensing in recent years,breaking the conventional fixed pattern of fluorescence detection as the output carrier,and many cutting-edge and innovative works have emerged.The biosensing technology has given a favorable platform for giving full play to and amplifying the molecular diagnostic capability of CRISPR,breaking the inertia of using fluorescent reporter molecules as indicator signals.And the CRISPR technology has brought new enhancements and breakthroughs to the application scope and detection level of the biosensing platform,which complement each other.Here,we create a novel sensing platform termed as“CRISPR-FET” for accelerated,unamplified detection of SARS-CoV-2.Unlike previous CRISPR-based assays,we constructed a gold nanoparticle（AuNP）-decorated graphene field-effect transistor sensor and modified it with reporter of CRISPR/Cas13 system to induce an on-chip CRISPR reaction.Through this ingenious incorporation,SARS-CoV-2 ORF1 ab and N gene were accurately detected with high sensitivity and single-base specificity.What is more,the excellent anti-interference capability was demonstrated by the detection of target RNA spiked into negative throat swabs.Importantly,we combined cr RNAs targeting ORF1 ab and N to achieve a 20-fold improvement of the sensitivity with a LOD of 940 copies/m L,which is non-inferior to RT-PCR but without PCR-related laborious operation.Under the dual-cr RNA strategy,the developed sensor successfully distinguished 10 COVID-19 patients from 10 healthy individuals.Of interest is that the sensor is expected to be a versatile platform for various RNA detection by altering the spacer sequence of cr RNA accordingly.Consequently,we believe that this powerful platform can be an attractive candidate for COVID-19 confirmation.