DNA Biosensor Research Articles

Electrochemical DNA-nano biosensor for the detection of Goserelin as anticancer drug using modified pencil graphite electrode.

Goserelin is an effective anticancer drug, but naturally causes several side effects. Hence the determination of this drug in biological samples, plays a key role in evaluating its effects and side effects. The current studies have concentrated on monitoring Goserelin using an easy and quick DNA biosensor for the first time. In this study, copper(II) oxide nanoparticles were created upon the surface of multiwalled carbon nanotubes (CuO/MWCNTs) as a conducting mediator. The modified pencil graphite electrode (ds-DNA/PA/CuO/MWCNTs/PGE) has been modified with the help of polyaniline (PA), ds-DNA, and CuO/MWCNTs nanocomposite. Additionally, the issue with the bio-electroanalytical guanine oxidation signal in relation to ds-DNA at the surface of PA/CuO/MWCNTs/PGE has been examined to determination Goserelin for the first time. It also, established a strong conductive condition to determination Goserelin in nanomolar concentration. Thus, Goserelin's determining, however, has a 0.21 nM detection limit and a 1.0 nM-110.0 µM linear dynamic range according to differential pulse voltammograms (DPV) of ds-DNA/PA/CuO/MWCNTs/PGE. Furthermore, the molecular docking investigation highlighted that Goserelin is able to bind ds-DNA preferentially and supported the findings of the experiments. The determining of Goserelin in real samples has been effectively accomplished in the last phase using ds-DNA/PA/CuO/MWCNTs/PGE.

Deep eutectic solvent-assisted synthesis of gold nanoflowers supported on glassy carbon electrode for DNA sensor application

Gold nanoflowers (AuNF) were synthesized on a glassy carbon electrode via a one-step, eco-friendly protocol in deep eutectic solvent (DES) of choline choloride and urea, called reline, for label-free detection of DNA hybridization. DES is eco-friendly, low-cost, biocompatible, and nontoxic, and it can be used as an electrolyte to synthesize nanomaterials by using the electrochemical method. In this protocol, highly branched and stable AuNFs were obtained without using any surfactants for DNA sensor application. The electrochemical performance of the AuNF-modified electrode was studied by cyclic voltammetry and electrochemical impedance spectroscopy. Under optimal conditions, the AuNF-based DNA biosensor exhibited a sensitivity of 294.9 Ω nM−1cm−2 and 218 μA nM−1cm−2 and a limit of detection (LOD) of 10−9 M. The remarkable sensitivity and low LOD could be attributed to the good conductivity of AuNFs for accelerating electron transfer, resulting in obvious signal amplification. The DNA biosensor showed good reproducibility (RSD <3.65 %) and acceptable stability and selectivity. Its excellent performance in DNA detection suggested that the proposed electrochemical DNA sensor has great application potential in clinical diagnosis.

Liquid crystal assisted optical DNA biosensor for the selective detection of bacterium Neisseria gonorrhoeae

ABSTRACT A highly selective and sensitive label-free liquid crystal (LC)-based optical DNA biosensor has been reported for the detection of bacterial disease. A sensing platform is constructed using nematic LC (4-cyano-4’−pentyl biphenyl) filled in a confined mesh area of transmission electron microscope (TEM) grid placed within a Polydimethylsiloxane (PDMS) well of a fixed dimension for the detection of bacterial infection caused by Neisseria gonorrhoeae. The optical response associated with LC orientation was investigated by using N. gonorrhoeae nucleotide-specific oligonucleotide probe interacting with both the synthetic complementary ssDNA as well as genomic DNA isolated from N. gonorrhoeae under polarised optical microscope. The grey scale area of the resultant POM images was quantised using Image J software and monitored as a function of DNA concentration. The LC-cell has been able to detect the genomic DNA of N. gonorrhoeae bacteria down to the lowest detectable concentration of 10 pM. The optical response studies of LC-biosensor with genomic DNAs isolated from different bacteria showed high specificity and selectivity exclusively for N. gonorrhoeae pathogen and its potential as a convenient, rapid, and reliable label-free detection for gonorrhoea disease.

Critical Issues on the Surface Functionalization of Plasmonic Au-Ag/TiO2 Thin Films with Thiolated Oligonucleotide-Based Biorecognition Elements.

This work reports on the surface functionalization of a nanomaterial supporting localized surface plasmon resonances (LSPRs) with (synthetic) thiolated oligonucleotide-based biorecognition elements, envisaging the development of selective LSPR-based DNA biosensors. The LSPR thin-film transducers are composed of noble metal nanoparticles (NPs) embedded in a TiO2 dielectric matrix, produced cost-effectively and sustainably by magnetron sputtering. The study focused on the immobilization kinetics of thiolated oligonucleotide probes as biorecognition elements, followed by the evaluation of hybridization events with the target probe. The interaction between the thiolated oligonucleotide probe and the transducer's surface was assessed by monitoring the LSPR signal with successive additions of probe solution through a microfluidic device. The device was specifically designed and fabricated for this work and adapted to a high-resolution LSPR spectroscopy system with portable characteristics. Benefiting from the synergetic characteristics of Ag and Au in the form of bimetallic nanoparticles, the Au-Ag/TiO2 thin film proved to be more sensitive to thiolated oligonucleotide binding events. Despite the successful surface functionalization with the biorecognition element, the detection of complementary oligonucleotides revealed electrostatic repulsion and steric hindrance, which hindered hybridization with the target oligonucleotide. This study points to an effect that is still poorly described in the literature and affects the design of LSPR biosensors based on nanoplasmonic thin films.

Inhibition of kinetic random-distribution in DNA Seesaw gates and biosensors for complete leakage prevention

DNA nanomaterials have a wide application prospect in biomedical field, among which DNA computers and biosensors based on Seesaw-based DNA circuit is considered to have the most development potential. However, the serious leakage of Seesaw-based DNA circuit prevented its further development and application. Moreover, the existing methods to suppress leakage can't achieve the ideal effect. Interestingly, we found a new source of leakage in Seesaw-based DNA circuit, which we think is the main reason why the previous methods to suppress leakage are not satisfactory. Therefore, based on this discovery, we use DNA triplex to design a new method to suppress the leakage of Seesaw-based DNA circuit. Its ingenious design makes it possible to perfectly suppress the leakage of all sources in Seesaw-based DNA circuit and ensure the normal output of the circuit. Based on this technology, we have constructed basic Seesaw module, AND gate, OR gate, secondary complex circuits and DNA detector. Experimental results show that we can increase the working range of the secondary Seesaw-based DNA circuit by five folds and keep its normal output signal above 90%, and we can improve the LOD of the Seesaw-based DNA detector to 1/11 of the traditional one(1.8pM). More importantly, we successfully developed a detector with adjustable detection range, which can theoretically achieve accurate detection in any concentration range. We believe the established triplex blocking strategy will greatly facilitate the most powerful Seesaw based DNA computers and biosensors, and further promote its application in biological systems.

Sequestration within peptide coacervates improves the fluorescence intensity, kinetics, and limits of detection of dye-based DNA biosensors

Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) – ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality.

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.

Sea Anemone-like Nanomachine Based on DNA Strand Displacement Composed of Three Boolean Logic Gates: Diversified Input for Intracellular Multitarget Detection.

Efficient and accurate acquisition of cellular biomolecular information is crucial for exploring cell fate, achieving early diagnosis, and the effective treatment of various diseases. However, current DNA biosensors are mostly limited to single-target detection, with few complex logic circuits for comprehensive analysis of three or more targets. Herein, we designed a sea anemone-like DNA nanomachine based on DNA strand displacement composed of three logic gates (YES-AND-YES) and delivered into the cells using gold nano bipyramid carriers. The AND gate activation depends on the trigger chain released by upstream DNA strand displacement reactions, while the output signal relies on the downstream DNAzyme structure. Under the influence of diverse inputs (including enzymes, miRNA, and metal ions), the interconnected logic gates simultaneously perform logical analysis on multiple targets, generating a unique output signal in the YES/NO format. This sensor can successfully distinguish healthy cells from tumor cells and can be further used for the diagnosis of different tumor cells, providing a promising platform for accurate cell-type identification.

Biosensors Precision in Healthcare: A Scientific Odyssey

Biosensors have been the beacon of precision in the ever-evolving landscape of health care and offer a profound impact on diagnostics and patient care. These devices are designed to detect specific biological markers and convert then into measurable signals, seamlessly blending biology with cutting-edge technology. The diverse tapestry of biosensors encompass enzyme based sensors, immuno-assays, photo-biosensors, tissue-based sensors, DNA biosensors and nano-biosensors. Enzyme-based biosensors showcase unparalleled catalytic precision exemplified by glucose biosensors. Glucose oxidase serves as the biological catalyst and converts glucose into gluconic acid and hydrogen peroxide. Electrodes of the sensor then transduce this reaction into measurable signals which enables the precise quantification of glucose levels in diabetes management. Similarly, immunoassays rely on the specificity of antigen-antibody interactions. Most widely used immunoassay, Enzyme-linked immunosorbent assay (ELISA) exemplify this category, which finds applications in a number of diagnostics and provide a window into immunological responses and diseases such as HIV. Advancing the field of biosensing are the photobiosensors which utilize diverse technologies such as fluorescence resonance energy transfer (FRET) to illuminate biological processes. Genetically encoded biosensors that employ proteins like green fluorescent protein (GFP), enable non-invasive monitoring of cellular dynamics and offer real-time insights. Another cateogory, tissue-based biosensors converge the technology with the physiological environments. For instance, implantable glucose sensors that continuously monitor blood glucose concentrations and provide real-time data for personalized treatment approaches. At the core of precision diagnostics lie DNA biosensors such as DNA microarrays. They offer parallel analysis of gen expression, influencing decisions in cancer diagnostics and pharmacogenomics. Nanobiosensors, represented by nanoparticle-based sensors, navigate the nano-frontiers with exceptional sensitivity. For example, quantum dots exhibit unique optical properties for sensitive detection. Likewise, gold nanoparticles based biosensors enable real-time monitoring of drug release in drug delivery systems and emphasize their role in precision medicine. As biosensors advance, ethical considerations come to the forefront. Issues such as data privacy, consent, and equitable access to these technologies demand careful attention. It becomes imperative to ensure that biosensors are developed and deployed responsibly with a keen awareness of potential societal implications in navigating the ethical landscape. In the symphony of precision that biosensors bring to healthcare, their types and applications reflect not only technological prowess but also the ethical responsibility inherent in their development and deployment. As these devices continue to evolve, the healthcare landscape stands on the brink of a new era, where precision and ethics intertwine for the betterment of patient outcomes.

Programmable Primer Switching for Regulating Enzymatic DNA Circuits.

Developing DNA strand displacement reactions (SDRs) offers crucial technical support for regulating artificial nucleic acid circuits and networks. More recently, enzymatic SDR-based DNA circuits have gained significant attention because of their modular design, high orthogonality signaling, and extremely fast reaction rates. Typical enzymatic SDRs are regulated by relatively long primers (20-30 nucleotides) that hybridize to form stable double-stranded structures, facilitating enzyme-initiated events. Implementing more flexible primer-based enzymatic SDR regulations remains challenging due to the lack of convenient and simple primer control mechanism, which consequently limits the development of enzymatic DNA circuits. In this study, we propose an approach, termed primer switching regulation, that implements programmable and flexible regulations of enzymatic circuits by introducing switchable wires into the enzymatic circuits. We applied this method to generate diverse enzymatic DNA circuits, including cascading, fan-in/fan-out, dual-rail, feed-forward, and feedback functions. Through this method, complex circuit functions can be implemented by just introducing additional switching wires without reconstructing the basic circuit frameworks. The method is experimentally demonstrated to provide flexible and programmable regulations to control enzymatic DNA circuits and has future applications in DNA computing, biosensing, and DNA storage.

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.

Rational design and synthesis of triazene-amonafide derivatives as novel potential antitumor agents causing oxidative damage towards DNA through intercalation mode

In this work, we rationally designed and synthesized two novel triazene-amonafide derivatives 2-(2-(diisopropylamino)ethyl)-5-(3,3-dimethyltriaz-1-en-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (D-11) and 5-(3,3-diethyltriaz-1-en-1-yl)-2-(2-(diisopropylamino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (D-12) as potential antitumor agents. The DNA damage induced by the intercalation mode of D-11 (D-12) towards DNA was electrochemically detected through the construction of efficient biosensors. The consecutive processes of reversible redox of naphthylimide ring and irreversible oxidation of triazene moiety were elucidated on the surface of glassy carbon electrode (GCE) by CV, SWV, and DPV methods. Electrochemical biosensors were obtained through the immobilization of ctDNA, G-quadruplexes, poly(dG), and poly(dA), respectively, on the clean surface of GCE. After the incubation of biosensors with D-11 or D-12, the peaks of dGuo and dAdo decreased prominently, and the peak of 8-oxoGua appeared at +0.50 V, suggesting that the interaction between D-11 (D-12) and DNA could result in the oxidative damage of guanine. Unexpected, the as-prepared DNA biosensor possessed satisfactory anti-interference property and good practicability in real samples. UV–vis and fluorescence spectra, and gel electrophoresis assays were employed to further confirm the intercalation mode of D-11 (D-12) towards DNA base pairs. Moreover, D-11 was proved to exhibit stronger anti-proliferation activity than mitionafide and amonafide against both A549 and HeLa cell lines.

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.

Label-Free DNA Hybridization Detection Using a Highly Sensitive Fiber Microcavity Biosensor.

A novel label-free optical fiber biosensor, based on a microcavity fiber Mach-Zehnder interferometer, was developed and practically demonstrated for DNA detection. The biosensor was fabricated using offset splicing standard communication single-mode fibers (SMFs). The light path of the sensor was influenced by the liquid sample in the offset open cavity. In the experiment, a high sensitivity of -17,905 nm/RIU was achieved in the refractive index (RI) measurement. On this basis, the probe DNA (pDNA) was immobilized onto the sensor's surface using APTES, enabling real-time monitoring of captured complementary DNA (cDNA) samples. The experimental results demonstrate that the biosensor exhibited a high sensitivity of 0.32 nm/fM and a limit of detection of 48.9 aM. Meanwhile, the sensor has highly repeatable and specific performance. This work reports an easy-to-manufacture, ultrasensitive, and label-free DNA biosensor, which has significant potential applications in medical diagnostics, bioengineering, gene identification, environmental science, and other biological fields.

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.

A biomimetic microfluidic chip based on the bubble filtration mechanism of stomatal pore membranes

Microfluidic chips hold vast potential for applications in fields such as DNA analysis, drug screening, cell culture and biosensing. However, microfluidic chips are susceptible to disruption and damage from bubbles....

CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ.

To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.

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.