Electrochemical DNA Biosensor Research Articles

Bioinspired superwettable electrode towards sensitive detection of myocardial infarction-specific miRNA

The development of a reliable and sensitive method for the detection of myocardial infarction (MI)-specific miRNAs is of great significance in the diagnosis and management of MI. Electrochemical biosensors have emerged as a promising tool for the detection of biomolecules due to their high sensitivity, selectivity, simplicity of operation, and miniaturization. In order to further improve the sensitivity and selectivity of DNA electrochemical biosensors and meet the requirements of microRNA detection in biological samples, we fabricated a patterned superwettable gold electrode (PS-Au electrode) by mimicking the superwettable micropatterns of the desert beetle. Combining this bioinspired electrode with a DSN-assisted target recycling amplification strategy, we achieved ultrasensitive detection of myocardial infarction-specific miRNA-483 ranging from 10 aM to 100 fM with a low detection limit of 5.32 aM for miRNA-483. The electrochemical biosensor based on PS-Au electrode and DSN-assisted target recycling amplification (PSE-DTRA-EB) has good stability and specificity. Our study provides a new method for the ultrasensitive detection of MI-specific miRNAs, which may have important clinical applications in the diagnosis and prognosis of MI.

Enhancing public health safety: Development and application of a MoS2@CNT-Chit electrochemical DNA biosensor for rapid and accurate detection of S. Typhi

This study introduces an advanced electrochemical biosensor that utilizes MoS2@CNT as an electrode material combined with a specific DNA probe to detect Salmonella Typhi rapidly and accurately. The sensor offers a broad detection range from 1.0 × 10−6 to 1.0 × 10−18 molL−1 and boasts an exceptionally low limit of detection (LOD) of 1.0 × 10−20 molL−1 for the target bacterium. It demonstrates a detection range from 1.0 × 104 to 1.0 × 1011 CFUml−1 in real samples, with a corresponding LOD of 1.0 × 104 CFUml−1. Rigorous testing against base mismatches and various bacterial strains confirms its specificity, ensuring reliable performance. Validated in real samples, the biosensor can accurately identify Salmonella Typhi in water and milk, achieving recoveries ranging from 92.95 % to 99.58 %. The exceptional performance of the biosensor is attributed to the MoS2@CNT electrode material and the specific DNA recognition probe, which enhance electron transfer and reduce steric impedance. These improvements contribute to the sensor's enhanced sensitivity and specificity, making it a significant advancement in public health safety by providing a rapid and accurate tool for detecting Salmonella Typhi in food samples.

Rapid monitoring and early warning of Phaeocystis globosa bloom based on an effective electrochemical biosensor

Harmful algal blooms (HABs) have posed a significant threat to human society and the ecological environment. In particular, the outbreak of Phaeocystis globosa (P. globosa) bloom could affect coasts nuclear power safety. Unfortunately, current ecological monitoring tools fail to dynamically detect the densities of solitary cells from P. globosa in the pre-outbreak phases, thus affecting early interventions. In the study, an effective electrochemical DNA biosensor was developed to serve the rapid and effective detection of P. globosa DNA through a specific DNA probe strategy. Especially, its good specificity and lower limit of detection (LOD, 17 pg/μL or 1063 cells/L) met the monitoring requirement of solitary-cell population change of P. globosa before the cyst formation (threshold: 1.0 × 107 cells/L), which is the key step in the algal bloom outbreaks and influences the outbreak cycle and scale. Furthermore, the accuracy of this electrochemical biosensor for the quantitative detection of solitary-cell P. globosa was confirmed by using the classical microscopic examination techniques (r = 0.981, P < 0.001). Moreover, its applicability was also validated by actual sample testing (r = 0.996, P < 0.001). Therefore, the novel technology offers great potential to improve dynamic detection and early warning of P. globosa bloom.

Label-free, background-free detection of nucleic acid with immobilization-free heterogeneous biosensor and one-pot hybridization chain reaction amplification

Although immobilization-free and label-free electrochemical DNA (E-DNA) biosensors have engaged tremendous interest due to their superior properties, such as easy operation, time-saving and cost-saving, most of them are fabricated in homogeneous modes and usually produce high background current. In the present work, we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction (HCR) for one-pot nucleic acid detection with zero background. The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins, resulting in the generation of HCR products with multibranched arms, which can be captured onto the electrode via π-π stacking interactions between multibranched arms and reduced graphene oxide (rGO). Prior to the incubation process with an electrode, two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes. Thus, an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized. This strategy also presents additional merits of simplicity and cheap cost, since probe immobilization, signal tag labeling, and multiple incubation processes are avoided. Therefore, the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.

Rapid, Point-of-Care Microwave Lysis and Electrochemical Detection of Clostridioides difficile Directly from Stool Samples.

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile.

Electrochemical DNA biosensor for HPV-16 detection based on novel carbon quantum dots/APTES composite nanofilm

Detection of high-risk human papillomavirus (HR-HPV) is critical for early warning and accurate screening of cervical disease in women. Electrochemical biosensors offer a promising analytical strategy that can circumvent the structural complexity of traditional optical components found in current bioassays. In this work, an innovative electrochemical biosensor for HPV-16 detection was developed, utilizing advanced nanomaterial technology and optimized DNA probe grafting processes. The self-assembled composite nanofilm was optimized by conducting an orthogonal analysis. The nanofilm exploited the unique advantages and outstanding performance of (3-aminopropyl)triethoxysilane (APTES) and carbon quantum dots (CQDs), which act as a “molecular bridge” between the biosensing interface and the electrode substrate. The electroactive molecule [Ru(NH3)6]3+ facilitated the highly sensitive signaling of the target DNA by interacting with the double-stranded DNA. Under optimal conditions, the developed electrochemical DNA biosensor exhibited high sensitivity in detecting HPV-16, with a limit of detection (LOD) of 0.73 fM, which showed exceptional potential to be used in widespread point-of-care (PoC) screening programs, opening up new opportunities for various applications.

A novel copper ion enhanced electrochemical DNA biosensor for the determination of epinephrine

A novel electrochemical biosensor was developed for the detection of epinephrine (EP) by immobilizing double-strand DNA (dsDNA) bound with copper ions on a gold electrode (Cu2+/dsDNA/MCH/AuE). The electrochemical behavior of EP at Cu2+/dsDNA/MCH/AuE was examined, and the results demonstrated a significant enhancement in the electrocatalytic oxidation peak current of EP due to the formation of a stable G-Cu(II)-G sandwich structure between Cu2+ and guanine at the modified electrode. The modification process of the electrode was characterized by scanning electron microscopy, infrared spectroscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry. A study on the effect of pH in phosphate buffer solution on the electrochemical oxidation of EP indicated that the catalytic oxidation process was pH-dependent. A plot of catalytic current versus EP concentration exhibited a dual-linear relationship within two ranges: 1.0–12.5 μM and 12.5–1000.0 μM, with correlation coefficients of 0.995 and 0.997, respectively. The limit of detection was determined to be 47 nM (S/N = 3). According to the calculated Hill coefficient (0.99), it can be concluded that the electrocatalytic process followed the Michaelis-Menten kinetic mechanism. The maximum catalytic current Im was 25 μA, while the apparent Michaelis-Menten constant Km was 1.425 mM. These findings indicated excellent electrocatalytic activity of the modified electrode towards oxidation of EP. The developed biosensor successfully detected EP in spiked mouse serum as well as epinephrine hydrochloride injection with high selectivity, sensitivity, stability, and accuracy.

An ultrasensitive electrochemical biosensor based on logic transcription circuit-activated DNAzyme amplifier for flap endonuclease 1 activity detection

The accurate detection of flap endonuclease 1 (FEN1) activity is crucial for early cancer diagnosis and therapy. Herein, we have established a “target-substrate double-enhanced” DNA electrochemical biosensor by integrating a cleavage-ligation logic transcription circuit-activated DNAzyme amplifier with the transition-metal carbides-gold nanoparticles (MXene-Au) composites for the accurate detection of FEN1 activity. The FEN1 can be translated into a stable transcription hairpin DNA (TH), incorporating the catalytic strand of the DNAzyme via the logic transcription circuit. Then, the TH hybridizes with the substrate strand of DNAzyme and the DNAzyme amplifier can be activated, thus producing a significant quantity of single-stranded DNA (sDNA) and achieving the efficient amplification of the FEN1. Finally, the sDNA loaded with electroactive substances is immobilized on the capture DNA-anchored MXene-Au modified electrode, significantly enhancing the electrochemical signal intensity for highly sensitive FEN1 activity detection. The excellent selectivity and sensitivity of the proposed electrochemical biosensor can facilitate the accurate detection of FEN1 activity in biological samples and aid in screening FEN1-releated drugs. Additionally, three-input concatenated logic gates (AND-AND, INHIBIT-AND, and AND-OR) are constructed for FEN1 activity detection. Particularly, the AND-AND gate achieves logic detection of FEN1 activity in complex samples. Consequently, this bidirectionally enhanced electrochemical biosensor offers a novel analytical method for FEN1 activity detection, showing substantial promise for early diagnosis and drug discovery of FEN1-related disease.

Immobilization-free and label-free electrochemical DNA biosensing based on target-stimulated release of redox reporter and its catalytic redox recycling

Herein, we demonstrate a simple, homogenous and label-free electrochemical biosensing system for sensitive nucleic acid detection based on target-responsive porous materials and nuclease-triggered target recycling amplification. The Fe(CN)63− reporter was firstly sealed into the pores of Fe3O4 nanoparticles by probe DNA. Target DNA recognition triggered the controllable release of Fe(CN)63− for the redox reaction with the electron mediator of methylene blue enriched in the dodecanethiol assembled electrode and thereby generating electrochemical signal. The exonuclease III (Exo III)-assisted target recycling and the catalytic redox recycling between Fe(CN)63− and methylene blue contributed for the enhanced signal response toward target recognition. The low detection limit toward target was obtained as 478 fM and 1.6 pM, respectively, by square wave voltammetry and cyclic voltammetry methods. It also possessed a well-discrimination ability toward mismatched strands and high tolerance to complex sample matrix. The coupling of bio-gated porous nanoparticles, nuclease-assisted target amplification and catalytic redox recycling afforded the sensing system with well-controllable signal responses, sensitive and selective DNA detection, and good stability, reusability and reproducibility. It thus opens a new avenue toward the development of simple but sensitive electrochemical biosensing platform.

Freestanding Nanofiber-Assembled Aptasensor for Precisely and Ultrafast Electrochemical Detection of Alzheimer's Disease Biomarkers.

Amyloid beta-protein (AβAβ) is a main hallmark of Alzheimer's disease (AD), and a low amount of Aβ protein accumulation appears to be a potential marker for AD. Here, an electrochemical DNA biosensor based on polyamide/polyaniline carbon nanotubes (PA/PANI-CNTs) is developed with the aim of diagnosing AD early using a simple, low-cost, and accessible method to rapidly detect Aβ42 in human blood. Electrospun PA nanofibers served as the skeleton for the successive in situ deposition of PANI and CNTs, which contribute both high conductivity and abundant binding sites for the Aβ42 aptamers. After the aptamers are immobilized, this aptasensor exhibits precise and specific detection of Aβ42 in human blood within only 4min with an extremely fast response rate, lower detection limit, and excellent linear detection range. These findings make a significant contribution to advancing the development of serum-based detection techniques for Aβ42, thereby paving the way for improved diagnostic capabilities in the field of AD.

An antifouling coating that enables electrochemical biosensing of MecA gene in complex samples

As an important emerging pollutant, antibiotic resistance genes (ARGs) monitoring is crucial to protect the ecological environment and public health, but its accurate detection is still a major challenge in complex environment samples. In this study, a robust antifouling electrochemical DNA biosensor (E-DNA biosensor) was proposed for highly sensitive and selective detection of the mecA gene in complex water samples. The antifouling coating of electrode consisted of a branched-shaped zwitterionic peptide and gold nanoparticles (CPEK-AuNPs). And, the PPPP linker of peptide between the anchoring and antifouling domains adopted a polyproline helix conformation. The CPEK-AuNPs-modified electrode interface with stable hydration layer prevented non-specific interactions while enhancing electron transfer to the electrode surface, resulting in high current response and outstanding antifouling performance. Coupled with synergistic isothermal strand displacement polymerase reaction (ISDPR), the proposed antifouling sensing strategy exhibited superb sensitivity for the mecA gene. The limit of detection (LOD) was as low as 16.67 fM (S/N = 3) in a broad linear range of 0.05–5000 pM, and a high specificity to resolve from even single base mismatch towards the complex water samples was obtained. Benefiting from the stable hydration layer, the as-prepared E-DNA biosensor preserved 91.8 % of the initial signal after 15 days of exposure in unprocessed wastewater samples. The excellent specificity, stability and reproducibility (RSD＜3%) of mecA gene detection indicated that the proposed sensing strategy with antifouling performance can be a versatile tool for accurate quantitative analysis of ARGs in complex environment samples.

Novel Determination of the Influence of Idarubicin upon DNA Chain Structure Using an Electrochemical DNA Biosensor by Voltammetry

Two sequences of DNA (single-strand DNA (ssDNA) and double-strand DNA (dsDNA)) modified disposable electrodes were designed to investigate the effect of antitumor agent drug idarubicine (IDA) on the DNA chain structure. The effect of IDA on DNA structure was analyzed by square wave voltammetry (SWV) depending on not only the guanine signal but also IDA oxidation response. The present study included the electrochemical investigation of IDA and the investigation of the biomolecular interaction between IDA and DNA. IDA was detected in buffer and urine using disposable pencil graphite electrodes (PGE) with SWV and cyclic voltammetry (CV)). The detection limit (LOD) of IDA in urine samples was 0.089 µg mL−1 with SWV under optimum conditions. For the biomolecular interaction of IDA and DNA, all electrochemical conditions, such as the concentration of DNA, concentration of IDA, interaction phase (at electrode surface/in solution phase), the interaction pH, and the interaction time were optimized. IDA interacted biomolecularly with DNA at the electrode surface and in solution phase using two methods. There is no previous electrochemical study performed on the interaction of IDA with ssDNA. Hence, the effect of IDA on both ssDNA and dsDNA was presented for the first time.

The development of electrochemical DNA biosensor based on woolen ball-shaped La3+/TiO2 nanostructure coating: Nanomolar detection of Vinorelbine

Although Vinorelbine (VOB) is a powerful anticancer medication, it inevitably has a number of adverse effects. Therefore, identifying this medication in biological samples is crucial for assessing both its effectiveness and negative effects. In this study, a new DNA biosensor was created to analyze VOB within biological samples. Moreover, the sensor has been created though the modification of a pencil graphite electrode (PEG) with polypyrrole (PP), a woolen ball-shaped La3+/TiO2 nanostructure (WB–S La3+/TiO2-NS) (enhancing the conductivity of electricity), along with ds-DNA (calf thymus) (ds-DNA/PP/WB-S La3+/TiO2-NS/PGE). Alterations in electrode signal has been then utilized to determine whether VOB is present. Under optimized condition, the differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques were adopted for electrochemical sensing of VOB. With a 0.37 nM detection limit, ds-DNA/PP/WB-S La3+/TiO2-NS/PGE demonstrated remarkable sensitivity for VOB at concentrations between 0.001 and 150.0 μM by DPV method. In addition, the calibration curve shows a linear range between 5.0 and 100.0 μM vanillin with a detection limit of about 0.76 μM by EIS method. Theoretical docking investigation was used to analyze the contacts among VOB and ds-DNA bases for future assessments, and the results supported the intercalation of VOB with guanine bases ds-DNA's intercalations. VOB was examined using the ds-DNA/PP/WB-S La3+/TiO2-NS/PGE method in actual samples.

Enhanced spin-polarization and detection limit in a spin-based optoelectrochemical DNA hybridization sensor induced by circularly polarized light

Enhanced spin polarization and L.O.D. in a spin-based electrochemical DNA biosensor using circularly polarized light.

A multi-channel electrochemical biosensor based on polyadenine tetrahedra for the detection of multiple drug resistance genes.

Antimicrobial resistance poses a serious threat to human health due to the high morbidity and mortality caused by drug-resistant microbial infections. Therefore, the development of rapid, sensitive and selective identification methods is key to improving the survival rate of patients. In this paper, a sandwich-type electrochemical DNA biosensor based on a polyadenine-DNA tetrahedron probe was constructed. The key experimental conditions were optimized, including the length of polyadenine, the concentration of the polyadenine DNA tetrahedron, the concentration of the signal probe and the hybridization time. At the same time, poly-avidin-HRP80 was used to enhance the electrochemical detection signal. Finally, excellent biosensor performance was achieved, and the detection limit for the synthetic DNA target was as low as 1 fM. In addition, we verified the practicability of the system by analyzing E. coli with the MCR-1 plasmid and realized multi-channel detection of the drug resistance genes MCR-1, blaNDM, blaKPC and blaOXA. With the ideal electrochemical interface, the polyA-based biosensor exhibits excellent stability, which provides powerful technical support for the rapid detection of antibiotic-resistant strains in the field.

A portable label-free electrochemical DNA biosensor for rapid detection of Salmonella Typhi.

A highly accurate, rapid, portable, and robust platform for detecting Salmonella enterica serovar Typhi (S. Typhi) is crucial for early-stage diagnosis of typhoid to avert and control the outbreaks of this pathogen, which threaten global public health. This study presents a proof-of-concept for our developed label-free electrochemical DNA biosensor system for S. Typhi detection, which employs a printed circuit board gold electrode (PCBGE), integrated with a portable potentiostat reader. Initially, the functionalized DNA biosensor and target detection were characterized using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods using a benchtop potentiostat. Interestingly, the newly developed DNA biosensor can identify target single-stranded DNA concentrations ranging from 10 nM to 20 μM, achieving a detection limit of 7.6 nM within a brief 5 minute timeframe. Under optimal detection conditions, the DNA biosensor exhibits remarkable selectivity, capable of distinguishing a single mismatch base pair from the target single-stranded DNA sequence. We then evaluated the feasibility of the developed DNA biosensor system as a diagnostic tool by detecting S. Typhi in 50 clinical samples using a portable potentiostat reader based on the DPV technique. Remarkably, the developed biosensor can distinctly distinguish between positive and negative samples, indicating that the miniaturised DNA biosensor system is practical for detecting S. Typhi in real biological samples. The developed DNA biosensor device in this work proves to be a promising point-of-care (POC) device for Salmonella detection due to its swift detection time, uncomplicated design, and streamlined workflow detection system.

Optimizing electrochemical DNA biosensors for the detection of avian infectious bronchitis virus

Optimizing electrochemical DNA biosensors for the detection of avian infectious bronchitis virus

Detection of high-risk HPV 16 genotypes in cervical cancers using isothermal DNA amplification with electrochemical genosensor

Cervical cancer emerges as the third most prevalent types of malignancy among women on a global scale. Cervical cancer is significantly associated with the persistent infection of human papillomavirus (HPV) type 16. The process of diagnosing is crucial in order to prevent the progression of a condition into a malignant state. The early detection of cervical cancer through initial stage screening is of the utmost significance in both the prevention and effective management of this disease. The present detection methodology is dependent on quantitative polymerase chain reaction (qPCR), which necessitates the use of a costly heat cycler instrument. In this study, we report the development of an electrochemical DNA biosensor integrated with an isothermal recombinase polymerase amplification (RPA) reaction for the detection and identification of the high-risk HPV-16 genotype. The electrochemical biosensor exhibited a high degree of specificity and sensitivity, as evidenced by its limit of detection (LOD) of 0.23 copies/μL of HPV-16 DNA. The validity of this electrochemical platform was confirmed through the analysis of 40 cervical tissues samples, and the findings were consistent with those obtained through polymerase chain reaction (PCR) testing. Our straightforward electrochemical detection technology and quick turnaround time at 75 min make the assay suitable for point-of-care testing in low-resource settings.

An attomolar-level electrochemical DNA biosensor based on target-triggered and entropy-driven catalytic amplification integrated with AuNPs@ZIF-8 nanocomposites for oral cancer overexpressed 1 detection

It is of great interest and necessity to develop a nonenzymatic, simple but highly sensitive biosensor for early diagnosis of oral cancer. Present here is an electrochemical DNA biosensor which integrates a target-triggered, entropy-driven, nonenzymatic and isothermal amplification strategy with gold nanoparticles/zeolitic imidazolate frameworks-8 (AuNPs@ZIF-8) nanocomposites for ultra-sensitive detection of oral cancer-related biomarker (ORAOV 1) in saliva. It is worth noting that the nuclease is not involved in the whole reaction process, which is simple and flexible in design only using a series of linear single-stranded DNA, avoiding undesired secondary structure interference. Meanwhile, due to the synergistic effect of AuNPs and ZIF-8, AuNPs@ZIF-8 nanocomposites display high stability, excellent electrical conductivity and exceptional electrocatalytic activity, further enhancing the electrochemical signal and avoiding labeling electrochemical signal probes. Experimental results demonstrate that this electrochemical DNA biosensor has a wide linear range (1 fM ∼1 nM), a low limit of detection (163 aM), excellent specificity, superior reproducibility and stability to ORAOV 1. More importantly, the actual application of the newly developed electrochemical biosensor is exemplified in human saliva with satisfactory recoveries. Therefore, the newly developed electrochemical biosensor has a broad application prospect in the nondestructive and early screening of oral cancer.

A Sensitive Electrochemical DNA Biosensor for Determination of Anti-Cancer Drug Gemcitabine Based on an AuNPs/MWCNTs/Carbon Paste Electrode

GMB is a common anticancer drug that needs careful monitoring because of its variable and toxic effects. Detecting GMB in biological samples can help adjust the dosage and enhance the treatment outcomes. In this study, a novel electrochemical sensor was developed based on a carbon paste electrode modified with single stranded DNA, gold nanoparticles and multi-walled carbon nanotubes. We characterized the unmodified (bare CPE) and modified (ss-DNA/AuNPs/MWCNTs/CPE) electrodes using scanning electron microscopy (SEM), EDX analysis and cyclic voltammetry (CV) techniques. The oxidation peak current was linearly proportional to the GMB concentration in two linear ranges: 1–10 μM and 10–50 μM, with a detection limit of 0.52 μM and a limit of quantification (LOQ) of 1.75 μM. The lower linear concentration range (1–10 μM) showed a sensitivity of 5.68 A.M−1.cm−2, while the higher range (10–50 μM) showed a sensitivity of 1.34 A.M−1.cm−2. We also investigated the repeatability, applicability and reproducibility of this method for human serum samples. We tested the selectivity and practical ability of the ss-DNA/AuNPs/MWCNTs/CPE for the determination of GMB in the presence of various interfering species and human serum samples. The results indicated that the ss-DNA/AuNPs/MWCNTs/CPE was a selective, reliable and accurate electrochemical sensor for GMB.