Label-free Detection Of DNA Hybridization Research Articles

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.

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.

Whispering Gallery Mode Microresonator Sensor Integrated in Exposed Core Fiber for Label-Free DNA Hybridization Detection

A novel optical fiber sensor using exposed-core fiber (ECF) to excite whispering gallery modes (WGMs) within a high refractive index barium titanate glass microsphere is proposed for achieving label-free real-time specific DNA detection. The microsphere is encapsulated into the exposed channel of the ECF allowing for physical stability while excited WGMs via the evanescent field. The structure enables the sensing area to be exposed to a large amount of aqueous solution and better contact with the sample to be analyzed. Using a transmission measurement, a high Q-factor in the order of 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sup> is obtained in an aqueous environment. To the best of our knowledge, this is the best Q-factor level obtained by the in fiber WGM sensor in an aqueous environment. Specific sequences of probe DNA are then bound to the surface of the microsphere so that specific detection of complementary DNA can be realized by tracking the wavelength shift the WGM resonance. Experimental results show a red shift of 5.6 pm for complementary DNA hybridization detection and less than 1 pm fluctuation for non-complementary DNA detection. The portable integrated sensor with miniature size has great potential for aqueous environmental sensing applications in the fields of diseases diagnostics, chemical analysis, and environmental protection.

In-fiber optofluidic michelson interferometer for detecting small volume and low concentration chemicals with a fiber ring cavity laser

An in-fiber optofluidic Michelson interferometer fabricated by femtosecond laser micromachining technology was presented. The interferometer structure was prepared by a special optical fiber with two cores and a microchannel. One core was connected to the core of a single-mode fiber (SMF) and functioned as a sensing arm on the microchannel inner wall, the other core worked as a reference arm and was connected to the optical waveguide introduced by femtosecond laser processing. By combining with a ring cavity laser, the detection limit (DL) of the interferometer could be improved. The laser sensor presented a high linear response with a refractive index (RI) sensitivity of 1039.77 nm/RIU and the calculated DL could reach as low as 1.675 × 10 −5 RIU. Then, the sensor measured different concentrations of vitamin C in tablets in the concentration range of 0–10 mg/mL with a detection limit of 0.06 mg/mL. Due to the advantages of optofluidic structure, the volume of the detection solution could be as low as few tens of nanoliters, and the time to replace and detect the solution only took a few seconds. In addition, the laser sensor realized label-free DNA hybridization detection through surface functionalization, and could detect 1 μM target DNA solution with high specificity. This sensing device had great potential for small volume and low concentration sample detection in the biochemical field. • An in-fiber optofluidic Michelson interferometer for detecting small volume and low concentration chemicals is presented. • The interferometer structure is prepared by a special microstructured optical fiber with two cores and a microchannel. • One core is connected with the SMF core as a sensing arm on the microchannel inner wall. • The other core is connected with an optical waveguide from the SMF core as a reference arm in the cladding. • Detection limit can reach as low as 0.06 mg/mL.

Chiral Sum Frequency Generation Spectroscopy Detects Double-Helix DNA at Interfaces.

Many DNA-based technologies involve the immobilization of DNA and therefore require a fundamental understanding of the DNA structure-function relationship at interfaces. We present three immobilization methods compatible with chiral sum frequency generation (SFG) spectroscopy at interfaces. They are the "anchor" method for covalently attaching DNA on a glass surface, the "island" method for dropcasting DNA on solid substrates, and the "buoy" method using a hydrocarbon moiety for localizing DNA at the air-water interface. Although SFG was previously used to probe DNA, the chiral and achiral SFG responses of single-stranded and double-stranded DNA have not been compared systemically. Using the three immobilization methods, we obtain the achiral and chiral C-H stretching spectra. The results introduce four potential applications of chiral SFG. First, chiral SFG gives null response from single-stranded DNA but prominent signals from double-stranded DNA, providing a simple binary readout for label-free detection of DNA hybridization. Second, with heterodyne detection, chiral SFG gives an opposite-signed spectral response useful for distinguishing native (D-) right-handed double helix from non-native (L-) left-handed double helix. Third, chiral SFG captures the aromatic C-H stretching modes of nucleobases that emerge upon hybridization, revealing the power of chiral SFG to probe highly localized molecular structures within DNA. Finally, chiral SFG is sensitive to macroscopic chirality but not local chiral centers and thus can detect not only canonical antiparallel double helix but also other DNA secondary structures, such as a poly-adenine parallel double helix. Our work benchmarks the SFG responses of DNA immobilized by the three distinct methods, building a basis for new chiral SFG applications to solve fundamental and biotechnological problems.

Real-time label-free detection of DNA hybridization using a functionalized graphene field effect transistor: a theoretical study.

Detection of DNA hybridization with high sensitivity and accuracy plays a major role in clinical diagnosis and treatment. Despite intense experimental studies of graphene field effect transistor as DNA hybridization detector, the mechanism of detection and changes in the electrical properties of the device is not investigated in detail. To this end, we have investigated an armchair graphene nanoribbon (AGNR) interconnected between gold electrodes as a detector of DNA hybridization. Using non-equilibrium Green’s function method and density functional theory, the effect of 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker, probe, and target DNA on the electrical properties of the device has been investigated at zero bias voltage. The results show that, after functionalization of AGNR with PBASE, the conductance of the device increases while functionalization with probe and target DNA leads to a decrease in conductance. The changes in the projected density of states on the AGNR and transmission around Fermi energy are the reason for the change in conductance of the system. In all cases, both charge transfer and electrostatic gating are responsible for the change in the electrical properties of the system. The results show that our device detects DNA hybridization with a sensitivity of 10% at zero bias voltage, and by applying a suitable gate voltage, it can show higher sensitivity.Graphical abstract

Gas-sensing and label-free detection of biomaterials employing multiple rings structured plasmonic nanosensor

In this paper, a refractive index sensor based on three circular rings coupled with a straight metal-insulator-metal (MIM) waveguide is presented. At near-infrared (NIR) spectrum, the spectral characteristics of the sensor are extensively analyzed using the finite element method (FEM). Moreover, the refractive index sensing property is comprehensively investigated by altering the design parameters. The results show a maximum sensitivity of 3573.3 nm/RIU with a figure of merit of 21.9, which is suitable for gas-sensing applications. The proposed sensor has also displayed prominent tolerance against manufacturing defects. Furthermore, the sensor proves to be highly sensitive for the label-free detection of blood components and DNA hybridization.

DNA Hybridization Measured with Graphene Transistor Arrays.

Arrays of field-effect transistors are fabricated from chemical vapor deposition grown graphene (GFETs) and label-free detection of DNA hybridization performed down to femtomolar concentrations. A process is developed for large-area graphene sheets, which includes a thin Al2 O3 layer, protecting the graphene from contamination during photolithographic patterning and a SiOx capping for biocompatibility. It enables fabrication of high-quality transistor arrays, exhibiting stable close-to-zero Dirac point voltages under ambient conditions. Passivation of the as-fabricated chip with a layer composed of two different oxides avoids direct electrochemical contact between the DNA solutions and the graphene layer during hybridization detection. DNA probe molecules are electrostatically immobilized via poly-l-lysine coating of the chip surface. Adsorption of this positively charged polymer induces a positive shift of the Dirac point and subsequent immobilization of negatively charged DNA probes induces a negative shift. Spatially resolved hybridization of DNA sequences is performed on the GFET arrays. End-point as well as real-time in situ measurements of hybridization are achieved. A detection limit of 10 fm is observed for hybridization of 20-nucleotide DNA targets. Typical voltage signals are around 100mV and spurious drifts below 1mV per hour.

Label-Free Electrochemical Detection of DNA Hybridization: A Method for COVID-19 Diagnosis.

This paper presents label-free electrochemical transduction as a suitable scheme for COVID-19-specific viral RNA/c-DNA detection, with an aim to facilitate point of care diagnosis. In lieu of this, we discuss the proposed electrochemical biosensing scheme, based on electrodeposited gold nanoparticles as the transducing elements. Specific to this approach, here, the protocols associated with the immobilization of the single-stranded probe nucleotide on to the biosensor, have also been laid out. This paper also discusses the methods of electrochemical analysis, to be used for data acquisition and subsequent calibration, in relation to target analyte detection. Towards facilitating portable diagnosis, development of miniaturized sensors and their integration with readout units have also been discussed.

Acetic Acid and Ammonium Persulfate Pre-Treated Copper Foil for the Improvement of Graphene Quality, Sensitivity and Specificity of Hall Effect Label-Free DNA Hybridization Detection.

The capability of graphene-based biosensors used to detect biomolecules, such as DNA and cancer marker, is enormously affected by the quality of graphene. In this work, high quality and cleanness graphene were obtained by CVD based on acetic acid (AA) and ammonium persulfate (AP) pretreated copper foil substrate. Hall effect devices were made by three kinds of graphene which were fabricated by CVD using no-treated copper foil, AA pre-treated copper foil and AP pre-treated copper foil. Hall effect devices made of AA pre-treated copper foil CVD graphene and AP pre-treated copper foil CVD graphene can both enhance the sensitivity of graphene-based biosensors for DNA recognition, but the AA pre-treated copper foil CVD graphene improves more (≈4 times). This may be related to the secondary oxidation of AP pre-treated copper foil in the air due to the strong corrosion of ammonium persulfate, which leads to the quality decrease of graphene comparing to acetic acid. Our research provides an efficient method to improve the sensitivity of graphene-based biosensors for DNA recognition and investigates an effect of copper foil oxidation on the growth graphene.

A CMOS Electrochemical Biochip With 32$\times$ 32 Three-Electrode Voltammetry Pixels

A CMOS-integrated electrochemical biochip for high-performance molecular testing is presented. The system includes an array of 32 $\times $ 32 three-electrode voltammetry pixels and on-chip temperature control between 25 and 95 °C. Each 100 $\mu \text{m}\,\times 100\,\,\mu \text{m}$ pixel includes a CMOS-compatible and chemo-stable amorphous carbon electrode transducer connected to in-pixel $\Sigma \Delta $ current detection circuitry with 280 fA rms input-referred noise (0.1–20 Hz bandwidth) and 93 dB dynamic range (DR). Array-based DNA detection assays are implemented, and successful DNA melt-analysis and real-time label-free DNA hybridization detection are reported.

Corrections to “Label-Free Detection of DNA Hybridization Utilizing Dual S-Tapered Thin-Core Fiber Interferometer”

Presents corrections to above named paper.

Label-Free Electrochemical Detection of DNA Hybridization Related to Anthrax Lethal Factor by using Carbon Nanotube Modified Sensors

Background: Anthrax Lethal Factor (ANT) is the dominant virulence factor produced by B. anthracis and is the major cause of death of infected animals. In this paper, pencil graphite electrodes GE were modified with single-walled and multi-walled carbon nanotubes (CNTs) for the detection of hybridization related to the ANT DNA for the first time in the literature. Methods: The electrochemical monitoring of label-free DNA hybridization related to ANT DNA was explored using both SCNT and MCNT modified PGEs with differential pulse voltammetry (DPV). The performance characteristics of ANT-DNA hybridization on disposable GEs were explored by measuring the guanine signal in terms of optimum analytical conditions; the concentration of SCNT and MCNT, the concentrations of probe and target, and also the hybridization time. Under the optimum conditions, the selectivity of probe modified electrodes was tested and the detection limit was calculated. Results: The selectivity of ANT probes immobilized onto MCNT-GEs was tested in the presence of hybridization of probe with NC no response was observed and with MM, smaller responses were observed in comparison to full-match DNA hybridization case. Even though there are unwanted substituents in the mixture samples containing both the target and NC in the ratio 1:1 and both the target and MM in the ratio 1:1, it has been found that ANT probe immobilized CNT modified graphite sensor can also select its target by resulting with 20.9% decreased response in comparison to the one measured in the case of full-match DNA hybridization case Therefore, it was concluded that the detection of direct DNA hybridization was performed by using MCNT-GEs with an acceptable selectivity. Conclusion: Disposable SCNT/MCNT modified GEs bring some important advantages to our assay including easy use, cost-effectiveness and giving a response in a shorter time compared to unmodified PGE, carbon paste electrode and glassy carbon electrode developed for electrochemical monitoring of DNA hybridization. Consequently, the detection of DNA hybridization related to the ANT DNA by MCNT modified sensors was performed by using lower CNT, probe and target concentrations, in a shorter hybridization time and resulting in a lower detection limit according to the SCNT modified sensors. In conclusion, MCNT modified sensors can yield the possibilities leading to the development of nucleic acid sensors platforms for the improvement of fast and cost-effective detection systems with respect to DNA chip technology.

Suspended CNT-Based FET sensor for ultrasensitive and label-free detection of DNA hybridization

A suspended carbon nanotube (SCNT)-based field effective transistor (SCNT-FET), which was fabricated by utilizing the surface tension of liquid silver to suspend a CNT between two Pd electrodes, was proposed for the detection of DNA hybridization. Benefits from the separation between the CNT and the substrates could be observed; namely, the conductivity of a SCNT-FET was much higher (two orders of magnitude) than that of a FET based on an unsuspended CNT and about 50% sensing surface of CNT was freed from substrate. The Slater-Koster tight-binding method was adopted for geometry optimization and transport property calculation of the SCNT bound with DNA. The result showed that the conductance (G = 1/R) of the SCNT decreased in order with the binding of single-stranded DNA (SSDNA, probe DNA) and double-stranded DNA (DSDNA) and that the ability of DSDNA to weaken the conductivity of the SCNT was several times higher than that of SSDNA. SEM and Raman spectroscopy were used to demonstrate that DNA could be bound successfully onto the SCNT using a 1-pyrenebutanoic acid succinimidyl ester (PBASE) as a linkage. Ultra-high sensitivity detection of DNA [with a limit of detection (LOD) as low as 10 aM] was obtained using such an SCNT-FET, which showed a lower value than that of a previously reported FET DNA biosensor whose sensing materials were in direct contact with the substrate.

Wafer-scale HfO2 encapsulated silicon nanowire field effect transistor for efficient label-free DNA hybridization detection in dry environment

AbatractSilicon nanowire (SiNW) charge based biosensors are attractive for DNA sensing applications due to their compactness and large surface-to-volume ratio. Small feature size, low production cost, repeatability, high sensitivity and selectivity are some of the key requirements for biosensors. The most common e-beam manufacturing method employed to manufacture sub-nm SiNWs is both cost and time intensive. Therefore, we propose a highly reproducible CMOS industry grade low-cost process to fabricate SiNW-based field effect transistors on 4″-wafers. The 60 nm wide SiNWs reported in this paper are fabricated using the sidewall transfer lithography process which is a self-aligned-double-patterning I-line lithography process that also facilitates encapsulation of the SiNW surface with a thin HfO2 layer on which DNA probes are grafted to finalize the biosensors. Upon DNA hybridization, SiNW devices exhibit threshold voltage shift larger than the noise introduced by the exposition to saline solutions used for the bioprocesses. More specifically, based on a statistical analysis, we demonstrate that 85% of the tested devices exhibit a positive threshold voltage shift after DNA hybridization. These promising results make way for the monolithic integration of SiNW biosensors and CMOS circuitry to realize a point of care device which can offer reliable real time electrical readout.

Attomolar Label-Free Detection of DNA Hybridization with Electrolyte-Gated Graphene Field-Effect Transistors.

In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one.

A miniaturized electrochemical platform with an integrated PDMS reservoir for label-free DNA hybridization detection using nanostructured Au electrodes.

We report the fabrication and characterization of a miniaturized electrochemical platform for the label-free detection of DNA hybridization. The proposed platform is fabricated using microfabrication and electrodeposition techniques. Comprising a Ti working electrode with electrodeposited Au nanostructures, and Pt/Au pseudo-reference and counter electrodes, the device accounts for a limit of detection of 0.97 fM and a sensitivity of 20.78 (μA μM-1) cm-2 with respect to Dengue virus specific consensus primer detection in the range of 10 fM-1 μM. Here, the incorporation of nanostructured Au in the active sensing area not only enhances the current response by increasing the overall surface area, but it also facilitates facile probe DNA immobilization by gold-thiol self-assembly. We have used differential pulse voltammetry analysis in this study to monitor the changes in reaction kinetics with respect to target hybridization. Furthermore, the evaluation of reproducibility of the biosensor and its selectivity against interference has yielded acceptable outcomes. Additionally, in order to evaluate the system's selectivity, we have successfully distinguished PCR amplified wild type and mutant target DNAs corresponding to the BRCA1 specific gene. Here, the mutant and the wild type target DNAs differ by a two base deletion, and the fact that the system is able to differentiate even such minute dissimilarities under hybridization conditions is indicative of its superior performance.

Label-Free Detection of DNA Hybridization Utilizing Dual S-Tapered Thin-Core Fiber Interferometer

A thin-core-fiber-based (TCF-based) dual S-tapered fiber sensor for label-free DNA hybridization detection has been proposed and experimentally demonstrated. The proposed biosensor is constructed by splicing a TCF of about 645 μ m in length with two segments of single-mode fibers, and the two S-tapers are fabricated at the splicing joints by using a commercial fusion splicer. By functionalizing the fiber surface with monolayer poly-L-lysine and single-stranded DNA (ssDNA) probes, label-free detection of target ssDNA is achieved. The sensor functionalization and the hybridization processes for 1 nM target ssDNA are monitored through real-time analysis of the transmission spectral evolution. Owing to its compactness, low cost, fast response, real-time detection, and good specificity and repeatability, the proposed biosensor is expected to find potential applications in label-free biomolecule detection.

Label-free detection of live cancer cells and DNA hybridization using 3D multilayered plasmonic biosensor

Three-dimensional (3D) multilayered plasmonic nanostructures consisting of Au nanosquares on top of SU-8 nanopillars, Au asymmetrical nanostructures in the middle, and Au asymmetrical nanoholes at the bottom were fabricated through reversal nanoimprint technology. Compared with two-dimensional and quasi-3D plasmonic nanostructures, the 3D multilayered plasmonic nanostructures showed higher electromagnetic field intensity, longer plasmon decay length and larger plasmon sensing area, which are desirable for highly sensitive localized surface plasmonic resonance biosensors. The sensitivity and resonance peak wavelength of the 3D multilayered plasmonic nanostructures could be adjusted by varying the offset between the top and bottom SU-8 nanopillars from 31% to 56%, and the highest sensitivity of 382 and 442 nm/refractive index unit were observed for resonance peaks at 581 and 805 nm, respectively. Live lung cancer A549 cells with a low concentration of 5 × 103 cells ml−1 and a low sample volume of 2 μl could be detected by the 3D multilayered plasmonic nanostructures integrated in a microfluidic system. The 3D plasmonic biosensors also had the advantages of detecting DNA hybridization by capturing the complementary target DNA in the low concentration range of 10−14–10−7 M, and providing a large peak shift of 82 nm for capturing 10−7 M complementary target DNA without additional signal amplification.

In-situ ultrasensitive label-free DNA hybridization detection using optical fiber specklegram

In this work, we present a compact and ultrasensitive bio-molecule sensor based on fiber specklegram, achieving a DNA detection sensitivity of 10−16 M. To our knowledge, the new device is the most sensitive fiber-optic DNA sensor reported to date. Importantly, the well-known temperature cross-sensitivity issue in many fiber-optic devices has been entirely removed by the introduction of a reference fiber-optic channel embedded in microfluidic channels. In the experiment, we demonstrate label-free DNA hybridization detection at different concentrations, where normalized intensity inner product of the collected specklegrams are calculated and used for determining the DNA concentration. The result shows the exact ssDNA concentration can be precisely determined with a single experiment in 60 min. The new fiber sensor presents distinctive advantages over traditional bio-sensors in terms of sensitivity, precision and cost, which may find many practical applications in health and medicine, e.g., point-of-care testing and disease diagnosis.