Construction of Electrochemical Biosensors Based on the CRISPR/Cas12 System for Applications

The current major issue in improving detection sensitivity and selectivity is to design an electrochemical sensor that does not require PCR amplification for nucleic acid identification and measurement. Because of their great sensitivity, precision, and simplicity of downsizing, electrochemical biosensors have emerged as a research hotspot in the field of nucleic acid detection. The CRISPR/Cas12 system has emerged as a potent tool for nucleic acid detection due to its powerful cleavage activity and selectivity. Specific electrode changes combined with the CRISPR/Cas12 system can greatly improve the performance of electrochemical biosensors. In this study, the design concepts of electrochemical biosensors based on the CRISPR/Cas12 system and their application advancements in nucleic acid detection are discussed.

DNA framework-engineered electrochemical biosensors

Self-assembled DNA nanostructures have shown remarkable potential in the engineering of biosensing interfaces,which can improve the performance of various biosensors.In particular,by exploiting the structural rigidity and programmability of the framework nucleic acids with high precision,molecular recognition on the electrochemical biosensing interface has been significantly enhanced,leading to the development of highly sensitive and specific biosensors for nucleic acids,small molecules,proteins,and cells.In this review,we summarize recent advances in DNA framework-engineered biosensing interfaces and the application of corresponding electrochemical biosensors.

Paper-Based Electrochemical Biosensors for Point-of-Care Testing of Neurotransmitters

Abstract Neurotransmitters are important biological molecules related to several nervous system diseases(NSDs).Point-of-care test-ing(POCT)of neurotransmitters is of great importance in preclinical research and early diagnosis of NSDs.Among various POCT platforms,paper-based electrochemical biosensors have achieved great advances in detection of neurotransmitters,thus taking a significant role in POCT of neurotransmitters nowadays.This review gives an overview of the recent advances of paper-based electrochemical biosensors for POCT of neurotransmitters.We first introduce the types of neurotransmitters and biological sample sources mainly used for neurotransmitter detection.Second,we review the components and the traditional fabrication technologies for paper-based electrochemical POCT biosensors,and then the functional modification methods of biosensor surfaces and three-dimensional fabrication methods for further enhancement of their detection performance.Then,we list examples of paper-based electrochemical biosensors used for detecting different neurotransmitters in biologi-cal samples.Last,we give a conclusion and promising development direction of paper-based electrochemical biosensors for neurotransmitters detection.The purpose of this review is to introduce the paper-based electrochemical biosensors as an emerging technology for POCT of neurotransmitters,offering a reference for readers and researchers for early diagnosis of NSDs using POCT technologies.

NiCo_2O_4 Nano-/Microstructures as High-Performance Biosensors: A Review

Non-enzymatic biosensors based on mixed transition metal oxides are deemed as the most promising devices due to their high sensitivity,selectivity,wide concentration range,low detection limits,and excellent recyclability.Spinel NiCo2O4 mixed oxides have drawn considerable attention recently due to their outstanding advantages including large specific surface area,high permeability,short electron,and ion diffusion pathways.Because of the rapid development of non-enzyme biosensors,the current state of methods for synthesis of pure and composite/hybrid NiCo2P4 materials and their subsequent electrochemical biosensing applications are systematically and comprehensively reviewed herein.Comparative analysis reveals better electrochemical sensing of bioanalytes by one-dimensional and two-dimensional NiCo2O4 nano-/microstructures than other morphologies.Better biosensing efficiency of NiCo2O4 as compared to corresponding individual metal oxides,viz.NiO and Co3O4,is attributed to the close intrinsic-state redox couples of Ni3+/Ni2+(0.58 V/0.49 V) and Co3+/Co2+(0.53 V/0.51 V).Biosensing performance of NiCo2O4 is also significantly improved by making the composites of NiCo2O4 with conducting carbonaceous materials like graphene,reduced graphene oxide,carbon nanotubes(single and multi-walled),carbon nanofibers;conducting polymers like polypyrrole(PPy),polyaniline(PANI);metal oxides NiO,Co3O4,SnO2,MnO2;and metals like Au,Pd,etc.Various factors affecting the morphologies and biosensing parameters of the nano-/microstructured NiCo2O4 are also highlighted.Finally,some drawbacks and future perspectives related to this promising field are outlined.

Development of ratiometric DNA biosensors with improved accuracy,precision,and signal-to-noise ratio

Ratiometric DNA biosensors,which utilize DNA as the recognition element and integrate dual signals from diverse sensing platforms including fluorescence,electrochemistry,and surface-enhanced Raman scattering(SERS),have demonstrated remarkable proficiency in detecting a wide range of targets.In this review,we showcase the significant progress achieved by ratiometric biosensors.Firstly,ratiometric biosensors have made notable advancements in analyzing real samples.These include the analysis of pH values near cancer cells,quantification of miRNA in human cell lysates,detection of human telomerase RNA in cell extracts,and performing DNA logicgated in situ bioimaging on cell membranes.Secondly,excellent sensitivity has been attained through the utilization of effective amplification methods such as RCA,HCR,and CHA,among others.Thirdly,the construction of stable reference signals has resulted in significantly improved precision for ratiometric biosensors.This breakthrough has overcome matrix effects,enabling reliable detection in real samples with high selectivity.This review provides a comprehensive overview of recent advancements in strategies employed by ratiometric DNA biosensors.We present three types of biosensors based on distinct sensing platforms:fluorescent,electrochemical,and SERS biosensors.Additionally,we discuss future directions and primary challenges in the development of ratiometric DNA biosensors.

Electrochemical Sensor for Dopamine Detection Based on the CRISPR/Cas12 System

Dopamine is an important neurotransmitter and biomarker that is involved in many physiological processes in the body as well as the control of the central nervous system. Therefore, it is crucial to accurately monitor dopamine concentrations in organisms in order to comprehend their biological roles and make correct clinical diagnoses. In this work, we describe the development of an aptamer sensor utilizing gold electrodes and cyclic voltammetry. Using a self-assembly approach, a single-chain sulfhydrylated dopamine-specific aptamer was immobilized on the surface of a gold electrode to successfully create the aptamer sensor. Voltammetry was used to do a thorough electrochemical characterization in order to assess the sensor’s performance. According to the findings, the created electrochemical sensor demonstrated outstanding analytical capabilities for the detection of dopamine, including a wide linear response range, a very low detection limit, high sensitivity, and great selectivity. These characteristics make the sensor a novel approach for the quick and precise detection of dopamine, and it is anticipated that clinical diagnostics and biological research will use it extensively.

Sensitive detection of SARS-CoV-2 spike protein based on electrochemical impedance spectroscopy of Fe_(3)O_(4)@SiO_(2)–Au/GCE biosensor

Highly contagious COVID-19 disease is caused by a novel severe acute respiratory syndrome coronavirus 2(SARSCoV-2),which poses a serious threat to global public health.Therefore,the development of a fast and reliable method for the detection of SARS-CoV-2 is an urgent research need.The Fe3O4@SiO2–Au is enriched with a variety of functional groups,which can be used to fabricate a sensitive electrochemical biosensor by biofunctionalization with angiotensin-converting enzyme 2(ACE2).Accordingly,we developed a novel electrochemical sensor by chemically modifying a glassy carbon electrode(GCE)with Fe3O4@SiO2–Au nanocomposites(hereafter Fe_(3)O_(4)@SiO_(2)–Au/GCE)for the rapid detection of S-protein spiked SARS-CoV-2 by electrochemical impedance spectroscopy(EIS).The new electrochemical sensor has a low limit detection(viz.,4.78 pg/mL)and a wide linear dynamic range(viz.,0.1 ng/mL to 10μg/mL)for detecting the EIS response signal of S-protein.The robust Fe_(3)O_(4)@SiO_(2)–Au/GCE biosensor has high selectivity,stability,and reproducibility for the detection of Sprotein with good recovery of saliva samples.

DNA Nano-netting Intertexture on Carbon Electrodes

Native calf thymus double stranded DNA (ct-dsDNA) is successfully immobilized from solution onto carbon substrates by covalent linkages under an optimized deposition potential of 1 .8±0.3 V vs. 50 mmol/L NaCl-Ag/AgCl. The long chain DNA fabricates a layer of well conductive nano-netting intertexture, which is stable in pH 14 alkaline solution and in boiling water. The ct-dsDNA modified carbon fiber disk electrode shows two to three orders of magnitude enlarged electrode effective surface area and similarly enlarged voltammetric responses to Co(phen)33+ and dopamine. Thermal dissociated single stranded ct-DNA can also lead to similar result. This modified electrode will find wide applications in the fields of DNA-based electrochemical biosensors.

Sensitive electrochemical DNA sensor for the detection of HIV based on a polyaniline/graphene nanocomposite

A nanocomposite of polyaniline/graphene(PAN/GN)was prepared using reverse-phase polymerization.The nanocomposite material was dropcast onto a glassy carbon electrode(GCE).Then,a single-stranded DNA(ssDNA)probe for HIV-1 gene detection was immobilized on the modified electrode,and the negative charged phosphate backbone of the HIV-1 was bound to the modified electrode surface via p-p*stacking interactions.The hybridization between the ssDNA probe and the target HIV-1 formed doublestranded DNA(dsDNA),and the electron transfer resistance of the electrode was measured using impedimetric studies with a[Fe(CN)6]^(3-/4-)redox couple.Under the optimized experimental conditions,the change of the impedance value was linearly related to the logarithm of the concentration of HIV genes in the range from 5.0×10^(-16)M to 1.0×10^(-10)M(R=0.9930),and the HIV sensor exhibited a lower detection limit of 1.0×10^(-16)M(S/N=3).The results show that this biosensor presented wonderful selectivity,sensitivity and specificity for HIV-1 gene detection.Thus,this biosensor provides a new method for the detection of HIV gene fragments.

A boronate-modified renewable nanointerface for ultrasensitive electrochemical assay of cellulase activity

The saccharification of cellulosic biomass to produce biofuels and chemicals is one of the most promising industries for gree n-power production and sustainable development.Cellulase is the core component in the saccharification process.Simple and efficient assay method to determine cellulase activity in saccharification is thus highly required.In this work,a boronate-affinity surface based renewable and ultrasensitive electrochemical sensor for cellulase activity determination has been fabricated.Through bo ronate-sugar interaction,celluloses are attached to the electrode surface,forming the cellulose na nonetwork at the sensing interface.Cellulase degradation can lead to the variation of electrochemical impedance.Thus,electrochemical impedance signal can reflect the cellulase activity.Importantly,via fully utilizing the boronate-affinity chemistry that enables reversible fabrication of cellulose nanonetwork,a renewable sensing surface has been firstly constructed for cellulase activity assay.Thanks to interfacial diffusion process of electrochemical sensor,the product inhibitory effect in the cellulase activity assays can be circumvented.The proposed electrochemical sensor is ultrasensitive for label-free cellulase activity detection with a very simple fabrication process,showing great potential for activity screen of new enzymes in saccharification conversion.

pH-Responsive Magnetic I-Motif Container Coupled with DNA Walker for Construction of Dual-Signal Electrochemical Biosensor

Herein,a dual-signal electrochemical biosensor has been developed by self-assembly of pH-activatable i-motif probes on magnetic microparticles(MMPs)coupled with DNA walker for signal amplification.In this study,the cytosine(C)-rich single-stranded DNAs are hybridized with DNA initiators to obtain the long-nicked duplexes with repeated units,which are further captured on MMPs to form the magnetic i-motif containers.The resulting duplexes contain abundant G-C base pairs,thus providing extensive binding sites for doxorubicin(DOX).At acidic pH,the C-rich sequences are folded into i-motif structure,resulting in the release of DOX and walker initiators.In this case,the liberated DOX is adsorbed on gra-phene quantum dots-modified glassy carbon electrode viaπ-πinteraction,while the walker initiators as a moving part can catalyze the hybridization between MB-modified fueler DNA and tracker DNA on electrode,contributing to the generation of dual electrochemical signals induced by MB and DOX.Importantly,the magnetic separation can effectively reduce the background,achieving sensitive biosensing of pH ranging from 4.0 to 7.4 with excellent stability.Moreover,the proposed dual-signal electrochemical biosensor has been successfully applied for accurate monitoring of pH in human serum,which holds great potential in pH-dependent bioassays,especially in ultra-micro analysis for clinical applications.

Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples

In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry （CV）. The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry （SWV）. The biosensor was able to detect the known genotoxic compounds： 2-anthramine, acridine orange and 2- naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxiciw bioassay has confirmed the applicability of the method for real samoles.

Aptamer-induced in-situ growth of acetylcholinesterase-Cu_(3)(PO_(4))_(2)hybrid nanoflowers for electrochemical detection of organophosphorus inhibitors

Enzyme-inorganic hybrid nanoflowers(HNFs)have shown excellent sensing capabilities due to their large specific surface area as well as the simplicity and mildness of the preparation process.However,coupling HNFs to electrodes to fabricate a uniform and controllable enzymatic electrochemical sensing interface remains a challenge.Here,we proposed an aptamer-induced insitu fabrication strategy for preparing an HNF-based electrochemical sensor with ideal performance.Central to this strategy is the introduction of acetylcholinesterase(AChE)-specific binding aptamer(Apt),which induces the in-situ growth of AChE-copper phosphate(Cu_(3)(PO_(4))_(2))HNFs on the surface of carbon paper(CP).In addition,a dense gold nanoparticle(AuNP)layer was electrodeposited on the CP for anchoring Apt and further extending the electroactive surface area.The prepared AChECu_(3)(PO_(4))_(2)HNF/Apt/AuNP/CP biosensor exhibited a wide detection range from 1 to 107 pM for the four organophosphorus inhibitors,including isocarbophos,dichlorvos,methamidophos,and parathion,with detection limits down to 0.016,0.028,0.071,and 0.113 pM,respectively.With the reactivation of pralidoxime chloride,the electrode can still recover 98.1%of the response after five times of repeated use.In real sample detection,the biosensor achieved high recoveries from 96.45%to 100.13%.The detection target may be extendable to other AChE inhibitors(e.g.,drugs for Alzheimer’s disease).This study demonstrates for the first time the feasibility of using aptamers as an inducer to fabricate an electrochemical enzyme sensing interface in-situ.This strategy can be used to fabricate other enzyme-based biosensors and therefore has broader applications.

Gold nanoparticles/single-stranded DNA-reduced graphene oxide nanocomposites based electrochemical biosensor for highly sensitive detection of cholesterol

High density and uniform distribution of the gold nanoparticles functionalized single-stranded DNA modified reduced graphene oxide nanocomposites were obtained by non-covalent interaction.The positive gold nanoparticles prepared by phase inversion method exhibited good dimensional homogeneity and dispersibility,which could readily combine with single-stranded DNA modified reduced graphene oxide nanocomposites by electrostatic interactions.The modification of single-stranded DNA endowed the reduced graphene oxide with favorable biocompatibility and provided the preferable surface with negative charge for further assembling of gold nanoparticles to obtain gold nanoparticles/single-stranded DNA modified reduced graphene oxide nanocomposites with better conductivity,larger specific surface area,biocompatibility and electrocatalytic characteristics.The as-prepared nanocomposites were applied as substrates for the construction of cholesterol oxidase modified electrode and well realized the direct electron transfer between the enzyme and electrode.The modified gold nanoparticles could further catalyze the products of cholesterol oxidation catalyzed by cholesterol oxidase,which was beneficial to the enzyme-catalyzed reaction.The as-fabricated bioelectrode exhibited excellent electrocatalytic performance for the cholesterol with a linear range of 7.5–280.5μmol·L^(−1),a low detection limit of 2.1μmol·L^(−1),good stability and reproducibility.Moreover,the electrochemical biosensor showed good selectivity and acceptable accuracy for the detection of cholesterol in human serum samples.

An electrochemical biosensor based on DNA“nano-bridge”for amplified detection of exosomal microRNAs

Exosomal miRNAs,as potential biomarkers in liquid biopsy for cancer early diagnosis,have aroused widespread concern.Herein,an electrochemical biosensor based on DNA“nano-bridge”was designed and applied to detect exosomal microRNA-21(miR-21)derived from breast cancer cells.In brief,the target miR-21 can specifically open the hairpin probe 1(HP1)labeled on the gold electrode(GE)surface through strand displacement reaction.Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction(HCR)between two kinetically trapped hairpin probes:HP2 immobilized on the GE surface and biotin labeled HP3 in solution.Cascade HCR leads to the formation of DNA“nano-bridge”tethered to the GE surface with a great deal of“piers”.Upon addition of avidin-modified horseradish peroxidase(HRP),numerous HRP were bound to the formed“nano-bridge”through biotin-avidin interaction to arouse tremendous current signal.In theory,only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted.Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L,as well as little cross-hybridization even at the single-base-mismatched level.Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line.To our knowledge,this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection.In a word,the high sensitivity,selectivity,low cost make the proposed method hold great potential application for early point-of-care(POC)diagnostics of cancer.

Recent Progress on Highly Selective and Sensitive Electrochemical Aptamer-based Sensors

Highly selective,sensitive,and stable biosensors are essential for the molecular level understanding of many physiological activities and diseases.Electrochemical aptamer-based(E-AB)sensor is an appealing platform for measurement in biological system,attributing to the combined advantages of high selectivity of the aptamer and high sensitivity of electrochemical analysis.This review summarizes the latest development of E-AB sensors,focuses on the modification strategies used in the fabrication of sensors and the sensing strategies for analytes of different sizes in biological system,and then looks forward to the challenges and prospects of the future development of electrochemical aptamerbased sensors.

Direct Electrochemistry of Hemoglobin on Vertically Aligned Carbon Hybrid TiO2 Nanotubes and Its Highly Sensitive Biosensor Performance

The present work is focused on developing a novel biomaterial platform to achieve enhanced direct electron transfer （DET） of hemoprotein and higher biosensor performance on vertically aligned carbon hybrid TiO2 nanotubes （C-TiO2 NTs）. Using a simple surfactant-assisted method, controllable hybridization of TiO2 NTs with conductive amorphous carbon species is realized. The obtained C-TiO2 NTs is ingeniously chosen to serve as an ideal ＂vessel＂ for protein immobilization and biosensor applications. Results show that the appropriate hybridization of C into TiO2 NTs leads to a much better conductivity of TiO2 NTs without destroying their preponderant tubular structures or damaging their excellent biocompatibility and hydrophilicity. When used in loading proteins, the C-TiO2 NTs can be used as a super vessel for rapid and substantive immobilization of hemoglobin （Hb）, with a large surface electroactive Hb coverage （I＊） of 3.3 × 10 9 mol·cm^-2. Enhanced DET of Hb is commendably observed on the constructed Hb/C-TiO2 NTs biosensor with a couple of well-defined redox peaks in a fast electron transfer process. The biosensor further exhibits fast response, high sensitivity and stability for the amperometric biosensing of H202 with the detection limit as low as 3.1 × 10^-8 mol/L.

Nano Au/TiO_2 hollow microsphere membranes for the improved sensitivity of detecting specific DNA sequences related to transgenes in transgenic plants

Gold nanoparticles (nano Au)/titanium dioxide (TiO2) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of nano Au and TiO2 microsphere was investigated with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored with EIS us-ing [Fe(CN)6]3-/4- as indicator. The sequence-specific DNA of the 35S promoter from cauliflower mosaic virus (CaMV35S) gene was detected with this DNA electrochemical sensor. The dynamic detection range was from 1.0×10-12 to 1.0×10-8 mol/L DNA and a detection limit of 2.3×10-13 mol/L could be ob-tained. The polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase (NOS) gene from the real sample of a kind of transgenic soybean was also satisfactorily detected.

Polypyrrole Hollow Nanotubes Loaded with Au and Fe_(3)O_(4) Nanoparticles for Simultaneous Determination of Ascorbic Acid,Dopamine,and Uric Acid

An ultrasensitive electrochemical biosensor was fabricated for electroanalytical determination of ascorbic acid(AA),dopamine(DA)and uric acid(UA)individually and simultaneously based on polypyrrole hollow nanotubes loaded with Au and Fe_(3)O_(4) nanoparticles(NPs)uniformly(PPy@Au-Fe_(3)O_(4)).The PPy@Au-Fe_(3)O_(4) nanotubes were synthesized in one-pot using MoO_(3) nanorods as templates and the polymerization of Py,the formation of Au and Fe_(3)O_(4) NPs and the removel of MoO_(3) templates took place stimultaneously.Electrochemical studies reveal that PPy@Au-Fe_(3)O_(4) modified glassy carbon electrode(GCE)possesses excellent electrocatalytic activities toward the oxidation of AA,DA and UA.Their oxidation peak currents increase linearly in the concentration ranges of 1–2000µmol/L for AA,0.01–25 and 25–300µmol/L for DA and 0.1–300µmol/L for UA.Their detection limit values(S/N=3)were calculated as 0.45,0.0049,and 0.051µmol/L for AA,DA and UA in the individual detection.By changing the concentrations simultaneously,the calibration curves showed linearity to 1000,200,and 200µmol/L with detection limit of 0.39,0.0060,and 0.060µmol/L for AA,DA,and UA,respectively.Finally,the obtained biosensor was successfully applied to the detection of AA,DA,and UA with satisfactory results on actual samples.

An enzyme-free glucose sensing device based on TiO2 nanorod array photoelectric catalysis

A simple one-step hydrothermal method is used to prepare an enzyme-free photoelectric combined glucose sensor based on TiO_(2)NRs/FTO with low cost,sample two-electrode,and excellent detection.Under 380 nm light(0.5 mW cm^(−2))irradiation and a positive voltage,holes are accumulated on TiO_(2)NRs surface,catalyzing glucose and forming a photocurrent without the need for an enzyme,such as Glucose oxidase(GOx).The designed sensor exhibits high sensitivity(about 0.96μA mM^(−1)cm^(−2),without GOx)and excellent linear relationship in the glucose concentration range of 5–15 mML^(−1).The prepared glucose sensor performs better with a sensitivity of 1.48μA mM^(−1)cm^(−2)when a certain amount of GOx is mixed in the detected solution.In addition,the sensor has excellent anti-interference resistance to non-reducing chitosan and reducing ascorbic acid with short response time(less than 5 s);thus,it can be used in quick detection with a double electrode system.This sensing device has the advantages of simple fabrication,easy storage,and reusability;therefore,it can be very promising in the portable and rapid monitoring of human blood glucose levels.