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Abstract The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3−/4− on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

The creation of large information molecules may have played an essential role in the origins of life. In this study, we conducted slow freeze‐thaw (F/T) experiments to test the possibility of enhanced hybridization between the complementary sticky ends attached to kilobase‐sized DNA fragments at sub‐nanomolar concentrations. DNA fragments of 2‐ and 3‐kilobase pairs (kbp) with 50‐base complementary sticky ends that can form 5 kbp‐sized hybridization products were mixed. While simple incubation provided little hybridization product, significantly effective hybridization was observed after freezing and thawing at a controlled time rate (<0.3 K min−1), even with small DNA concentrations (<1 nM). Furthermore, slow thawing had a more effect on hybridization than slow freezing. The reaction efficiency was reduced by rapid thawing instead of slow thawing, suggesting that the eutectic phase concentration played an important role in hybridization. A slow F/T cycle was effective even for the hybridization reaction between two 10 kbp DNA fragments, which yielded a 20 kbp product at sub‐nanomolar concentrations. Repeating the slow F/T cycle significantly improved the reaction efficiency. The possible role of the F/T cycles in early Earth environments is discussed here. [ABSTRACT FROM AUTHOR]

In the present work, a novel electrochemical DNA sensor was designed to detect L. monocytogenes. Two different gene fragments were selected for the sensor design. One is a 702 bp long fragment of the hlyA gene, encoding the synthesis of listeriolysin O toxin, which is unique only to pathogenic strains of L. monocytogenes and is essential for pathogenicity. The other is a 209 bp long fragment of the 16 S RNA gene found in all species of the Listeria genus. As the working electrode, the pencil graphite electrode was modified in various ways (activated or covered with polypyrrole), and six different combinations were constituted using three types of the modified working electrode and two different gene fragments. The developed system is based on differential pulse voltammetric transduction of guanine oxidation after hybridization between the selected gene fragment (38 µg/mL) and the selected fragment-specific inosine-modified probe (1.8 µmol/L) immobilized on a pencil graphite electrode surface. The comparison of all combinations demonstrates that the best results are obtained with the combination formed from a polypyrrole-coated pencil graphite electrode (prepared at 2 scans) and 702 bp fragment of the hlyA gene. The analysis time is less than 1 hour, and the necessary DNA concentrations for the analysis have been determined as 8.2 × 10−11 M DNA and 2.7 × 10−10 M DNA respectively, for the hlyA gene and 16 S RNA gene. [ABSTRACT FROM AUTHOR]

The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3−/4− on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease. [ABSTRACT FROM AUTHOR]

Simultaneous measurement of human immunodeficiency virus (HIV) genome DNA hybridization and the DNA melting temperature in a prism-based surface plasmon resonance (SPR) biosensor is modeled theoretically using a simple dual-channel construction. The proposed sensor consists of a BK7 prism coated with silver as a plasmonic material. The metal surface is divided into two channels to detect medium refractive index (RI) and temperature. One half is covered with zinc selenide (ZnSe) semiconductor to enhance the hybridization detection sensitivity and to protect silver from oxidation. The other half is covered with polydimethylsiloxane (PDMS) polymer to detect the temperature variations. The proposed sensor is optimized numerically, and the optimum structure provides an excellent sensitivity of 208 deg/RIU, thanks to the use of the ZnSe layer, which is greater than double the reported dual-channel prism-based sensor in thickness. The polymer channel shows high sensitivity to the temperature variations of − 0.125 deg/°C, which is nearly 10 times the response of the RI channel to temperature variations. The data obtained from the polymer channel is used to compensate for the thermal perturbations of the sensing medium RI, and at the same time, to monitor the increments of the temperature in order to avoid reaching the DNA melting temperature. A mathematical expression is provided to consider the effect of the temperature variations on the RI of the sensing medium to get a better accurate detection process. The DNA hybridization detection of HIV is theoretically discussed in detail starting from the preparation of the sensing medium with the different ingredients until the hybridization between probe and complementary target DNA (ct-DNA) molecules. [ABSTRACT FROM AUTHOR]

Graphene nanoribbon (GNR) is a carbon nanomaterial with strikingly distinct characteristics compared to graphene. GNRs possess high aspect ratio, variable band gap, high surface area and high density of reactive edge states which make it an emerging flat molecular platform alternative to graphene. GNR is not much explored in sensing applications compared to graphene. Here, for the first time, we are reporting the application of GNRs, derived from multiwalled carbon nanotube (MWCNT), as an efficient flat molecular platform for hosting and biosensing of Human immune deficiency virus-1 (HIV-1) gene. For the construction of HIV-1 sensor, GNRs were synthesized by unzipping of MWCNTs and deposited onto glassy carbon electrode (GCE) followed by the immobilization of HIV-1 probe (ssDNA) onto the resulting GCE/GNR platform to obtain GCE/GNR/ssDNA sensor. Subsequently, the sensor was employed in the analysis of target gene (cDNA) by measuring the voltammetric response generated from hybridization between ssDNA and cDNA. Differential pulse voltammetry (DPV) responses recorded at different target gene concentrations displayed perfect linear correlation (R2 = 0.991). The sensor exhibited low detection limit (2.3 aM), low quantification limit (7.67 aM) and high sensitivity (5.5 μA aM−1 cm−2) which is possibly due to synergic effects of GNRs. Sensor exhibited acceptable stability, repeatability and displayed good selectivity when tested with mismatched target DNA sequences. Further, sensor’s diagnostic applicability was verified by spiking target gene in human blood sample which showed high recovery (4 %) indicating its practical utility in the electroanalysis of HIV-1 gene in human blood samples.

Wafer-scale nanoribbon field-effect transistor (FET) biosensors fabricated by straightforward top-down processes are demonstrated as sensing platforms with high sensitivity to a broad range of biological targets. Nanoribbons with 350 nm widths (700 nm pitch) were patterned by chemical lift-off lithography using high-throughput, low-cost commercial digital versatile disks (DVDs) as masters. Lift-off lithography was also used to pattern ribbons with 2 μm or 20 μm widths (4 or 40 μm pitches, respectively) using masters fabricated by photolithography. For all widths, highly aligned, quasi-one-dimensional (1D) ribbon arrays were produced over centimeter length scales by sputtering to deposit 20 nm thin-film In2O3 as the semiconductor. Compared to 20 μm wide microribbons, FET sensors with 350 nm wide nanoribbons showed higher sensitivity to pH over a broad range (pH 5 to 10). Nanoribbon FETs functionalized with a serotonin-specific aptamer demonstrated larger responses to equimolar serotonin in high ionic strength buffer than those of microribbon FETs. Field-effect transistors with 350 nm wide nanoribbons functionalized with single-stranded DNA showed greater sensitivity to detecting complementary DNA hybridization vs 20 μm microribbon FETs. In all, we illustrate facile fabrication and use of large-area, uniform In2O3 nanoribbon FETs for ion, small-molecule, and oligonucleotide detection where higher surface-to-volume ratios translate to better detection sensitivities.

Background: Over the past 10 years, the clinical importance of opportunistic bacteria of the order Actinomycetales has increased significantly. While many problems for the Mycobacterium tuberculosis complex have been solved, for nontuberculous mycobacteria, some questions remain open. These pathogens have a number of structural features that allow them to persist in the external environment for a long time. Methods: The main inclusion criteria were cultural characteristics in assessing the growth of microorganisms on solid egg media. If nontuberculous mycobacteria (NTM) growth was detected, identification signs were carried out using the DNA hybridization method. Subsequently, these cultures were identified using the matrix-assisted laser desorption/ionization--time-of-flight (MALDI-ToF) mass spectrometry method. In case of obtaining unacceptable results of identification from primary inoculations, re-identification to obtain pure cultures was carried out after transferring the material from primary media to agar media: 5% blood agar and universal chromogenic medium. When re-identifying isolated cultures using MALDI-ToF mass spectrometry, all isolated cultures were analyzed, regardless of whether they belonged to the NTM group or not. Results: DNA hybridization, which accounted for 59.5% of the total number of cultures included in the study, performed species identification of 188 strains. Using MALDI-ToF mass spectrometry, 345 strains were identified. Conclusion: The use of methods based on DNA hybridization makes it possible to identify quite accurately some of the most common NTM species. MALDI-ToF mass spectrometry is an important technique to allow species identification of most Actinomycetales. However, algorithms to standardize methods for their isolation from clinical material are needed. [ABSTRACT FROM AUTHOR]

Milk is a widely consumed product, and its adulteration is not only common but also very dangerous. This study aimed to develop a biosensor for the detection of milk adulteration using DNA hybridization coupled with an electrochemical device. The advantages of biosensors over traditional laboratory methods, such as their speed, ease of use, and cost-effectiveness, are combined with the sensitivity of DNA hybridization. A capacitive biosensor was developed using interdigitated gold electrodes on paper substrate, which were modified with specific oligonucleotides for cow mitochondrial DNA that served as the biorecognition element. The methodology relies on the measurement of changes in capacitance due to DNA hybridization. Preliminary results are presented, showing the ability of this biosensor to detect bovine DNA in goat milk with high sensitivity and specificity. The results show that this biosensor has the potential to be a low-cost, easy-to-perform, and fast method for the detection of milk adulteration. This biosensor technology is a promising development for the detection of milk adulteration and can help to ensure the safety and quality of milk products. [ABSTRACT FROM AUTHOR]

Background: Over the past 10 years, the clinical importance of opportunistic bacteria of the order Actinomycetales has increased significantly. While many problems for the Mycobacterium tuberculosis complex have been solved, for nontuberculous mycobacteria, some questions remain open. These pathogens have a number of structural features that allow them to persist in the external environment for a long time. Methods: The main inclusion criteria were cultural characteristics in assessing the growth of microorganisms on solid egg media. If nontuberculous mycobacteria (NTM) growth was detected, identification signs were carried out using the DNA hybridization method. Subsequently, these cultures were identified using the matrix-assisted laser desorption/ionization–time-of-flight (MALDI-ToF) mass spectrometry method. In case of obtaining unacceptable results of identification from primary inoculations, re-identification to obtain pure cultures was carried out after transferring the material from primary media to agar media: 5% blood agar and universal chromogenic medium. When re-identifying isolated cultures using MALDI-ToF mass spectrometry, all isolated cultures were analyzed, regardless of whether they belonged to the NTM group or not. Results: DNA hybridization, which accounted for 59.5% of the total number of cultures included in the study, performed species identification of 188 strains. Using MALDI-ToF mass spectrometry, 345 strains were identified. Conclusion: The use of methods based on DNA hybridization makes it possible to identify quite accurately some of the most common NTM species. MALDI-ToF mass spectrometry is an important technique to allow species identification of most Actinomycetales. However, algorithms to standardize methods for their isolation from clinical material are needed.

For sensitive detection of the L-fuculokinase genome related to the Haemophilus influenzae (H. influenzae), this research work demonstrates the label-free electrochemical-based oligonucleotide genosensing assay relying on the performing hybridization process. To enhance the electrochemical responses, multiple electrochemical modifier-tagged agents were effectively utilized. For attaining this goal, NiCr-layered double hydroxide (NiCr LDH) has been synthesized and combined with biochar (BC) to create an efficient electrochemical signal amplifier that has been immobilized on the surface of the bare Au electrode. Low detection and quantification limits (LOD and LOQ) associated with the designed genosensing bio-platform to detect L-fuculokinase have been achieved to 6.14 fM and 11 fM, respectively. Moreover, the wide linear range of 0.1 to 1000 pM demonstrates the capability of the designed platform. Investigated were the 1-, 2-, and 3-base mismatched sequences, and the negative control samples clarified the high selectivity and better performance of the engineered assay. The values of 96.6–104% and 2.3–3.4% have been obtained for the recoveries and RSDs, respectively. Furthermore, the repeatability and reproducibility of the associated bio-assay have been studied. Consequently, the novel method is appropriate for rapidly and quantitatively detecting H. influenzae, and is considered a better candidate for advanced tests on biological samples such as urine samples. [ABSTRACT FROM AUTHOR]

Polymer-based photonic crystals (PhCs) were fabricated using nanoimprint lithography for use as DNA biosensors by immobilizing the probe DNA. Polymer-based PhCs can be applied to cost-effective and label-free DNA biosensors. Additionally, DNA hybridization can be detected using inexpensive and simple optical systems. In this study, probe DNA was immobilized on the PhC surface using electrostatic adsorption or covalent bonding, and the sensing performance was compared. Consequently, the PhC biosensor prepared using covalent bonding exhibited greater DNA-hybridization-induced reflection intensity changes than that prepared using electrostatic adsorption. Furthermore, discrimination of reflection intensity for target and mismatch DNA was investigated, which revealed that in contrast to the PhC biosensor prepared using the electrostatic adsorption of probe DNA, that prepared using covalent bonding discriminated target DNA at a concentration of 0.1 nM. [ABSTRACT FROM AUTHOR]

In this paper we present the characterization of 3D-printed nanocarbon electrodes (3DnCes) and their application in electrochemical enzyme-linked detection of DNA hybridization. The approach takes advantage of a facile procedure based on adsorption of target DNA on the electrode surface followed by hybridization with a biotinylated probe and binding of streptavidin–alkaline phosphatase conjugate. The alkaline phosphatase converts 1-naphthyl phosphate in the background electrolyte into electrochemically oxidizable 1-naphthol, which is subsequently detected using linear sweep voltammetry. The preparation, characterization, and analytical performance of the 3DnCes are reported. The results show the applicability of such 3DnCes in detection of target DNA hybridization specifically with the complementary biotinylated probe, and indicate the potential of 3D printed electrodes for use in various bioanalytical approaches.

Hybridization of surface‐bound DNA with complementary strands is the basis of many biotechnological applications. Herein, the structure of interfacial coatings between substrate and bound DNA is a crucial element for hybridization behavior. Herein, three reactive surfaces for constructing DNA‐sensing platforms, namely, plain gold films on silicon, poly(bisphenolA‐co‐epichlorohydrin) (PBAG) surfaces with a brush‐like bilayer structure, and dibenzocyclooctyne monolayers (both on glass), are compared. Fluorescence imaging is employed to survey the effect of coating structure and conformation on hybridization performance. To better understand the interfacial structural properties and chemistry of the coated films, atomic force microscopy, water contact angle measurements, and X‐ray photoelectron spectroscopy are employed to characterize the surface morphology. DNA probe microarrays are created on the different platforms via microchannel cantilever spotting, and their performance for hybridizing with the DNA counterparts is assessed. While all three platforms work reliable for DNA detection, a protein‐binding assay reveals that PBAG surfaces offer the highest hybridization efficiency among these approaches. The results of the present work have significant implications for comprehension of the interactions between the DNA hybridization efficiency and the physico‐chemical properties of surface coatings and can inform the fabrication of DNA sensors.

Quartz-crystal microbalance (QCM) is a technique that can measure nanogram-order masses. When a receptor is immobilized on the sensor surface of a QCM device, the device can detect chemical molecules captured by the mass change. Although QCM devices have been applied to biosensors that detect biomolecules without labels for biomolecular interaction analysis, most highly sensitive QCM devices are benchtop devices. We considered the fabrication of an IC card-sized QCM device that is both portable and battery-powered. Its miniaturization was achieved by repurposing electronic components and film batteries from smartphones and wearable devices. To demonstrate the applicability of the card-sized QCM device as a biosensor, DNA-detection experiments were performed. The card-sized QCM device could detect specific 10-mer DNA chains while discerning single-base differences with a sensitivity similar to that of a conventional benchtop device. The card-sized QCM device can be used in laboratories and in various other fields as a mass sensor. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (AuNPs) are popular labels for colorimetric detection of various analytes, involving proteins, nucleic acids, viruses, and whole cells because of their outstanding optical properties, inertness, and modification variability. In this work, we present an improved approach for enhancement of color intensity for DNA membrane microarrays based on seed-mediated growth of AuNP labels. Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microarrays. Then biotin is revealed by a streptavidin–AuNP conjugate followed by the detection of AuNPs. Optimization of seed-mediated enlargement of AuNPs by the reduction of tetrachloroauric acid with hydroxylamine made it possible to change the coloring of specific spots on the microarrays from pink to a more contrasting black with minor background staining. Mean size of the resulting AuNPs was four times larger than before the enhancement. Adjusting the pH of HAuCl4 solution to 3.5 and use of a large excess of hydroxylamine increased the signal/background ratio by several times. The method's applicability was demonstrated for quantification of a short oligonucleotide of 19 bases and full-length TEM-type β-lactamase genes of 860 bp responsible for the development of bacterial resistance against β-lactam antibiotics. Improved protocol for AuNP enlargement may be further transferred to any other membrane-based assays of nucleic acids with both instrumental and visual colorimetric detection. [ABSTRACT FROM AUTHOR]

Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications ‐ bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we used Gaussia luciferase (Gluc) as a donor and GO as a quencher and demonstrated its application in sensing of two target analytes, HIV‐1 DNA and IFN‐γ. We demonstrated that the incubation of Gluc conjugated HIV‐1 and IFN‐γ oligonucleotide probes with GO provided for monitoring of probe‐target interactions based on bioluminescence measurement in a solution phase sensing system. The limits of detection obtained for IFN‐γ and HIV‐1 DNA detection were 17 nM and 7.59 nM, respectively. Both sensing systems showed selectivity toward the target analyte. The detection of IFN‐γ in saliva matrix was demonstrated. The use of GO as a quencher provides for high sensitivity while maintaining the selectivity of designed probes to their respective targets. The use of GO as a quencher provides for an easy assay design and low cost, environmentally friendly reporter. [ABSTRACT FROM AUTHOR]

Telomeres, repeating TTAGGG sequences at the ends of eukaryotic chromosomes, increase genomic stability. Telomere shortening occurs not only during DNA replication associated with cell division but also under oxidative stress, where reactive oxygen species damage DNA. Therefore, changes in telomere length can be used to evaluate chronic cost or stress responses incurred by individuals. The phylogenetically unique Chondrichthyes are among the least-studied groups of marine vertebrates. Telomere data are limited and have only been reported in a few Chondrichthyes species. In this study, we measured telomere length and quantified oxidative stress and antioxidant power in 17 Chondrichthyes species whose telomere length has not been measured before. The presence of telomere sequences > 30 bp and lower values of oxidative stress were confirmed in most species. Average telomere length was not correlated with oxidative stress and antioxidant power in 15 species for which both measurements were available. It would be desirable in the future to elucidate the nature of telomeres in Chondrichthyes, and their direct relationship with reactive oxygen species. [ABSTRACT FROM AUTHOR]

A biosensor for detecting DNA hybridization via refractive index sensing has been demonstrated using a subwavelength grating (SWG) waveguide. Waveguide dimensions are selected by parametric optimizations to increase the mode-analyte overlap with considerations of typical silicon-on-insulator fabrications technology. DNA layer is added with a linker layer on silicon pillars of the SWG waveguide to optimize and detect the DNA hybridization. Some essential characteristics such as mode overlap factor, change in effective refractive index, waveguide sensitivity, and shift in resonance wavelength are computed for the different dimensional parameters of the SWG waveguide in DNA hybridization along with mode field intensity and normalized power using the finite element method. The DNA hybridization is shown by variation in normalized power of interacting light in cladding region of the waveguide for 0% to 100% fractional change of dsDNA and ssDNA. Waveguide sensitivity, shift in resonance wavelength, device sensitivity, and intrinsic limit of detection are obtained to ∼ 0.157, 7.01, 605 nm/RIU, and 1.30 × 10−4 RIU, respectively, for the optimized structure of SWG waveguide in sensing of DNA hybridization, which could be suitable aspects for detecting DNA hybridization. [ABSTRACT FROM AUTHOR]

• A dual-functional C-type fiber SPR biosensor for simultaneous DNA hybridization and temperature sensing is proposed. • Dual SPR effects were realized on the open microcavity by coating with a gold film and fillingwith thePDMS. • The sensor exhibits high sensitivity, with a response of 0.11 nm/nM for DNA molecules and −1.97 nm/℃ for temperature. • The limit of detection (LOD) for DNA molecules is 7.9 nM. • This compact sensor offers a promising approach for DNA detection with integrated temperature compensation. In this work, an integrated temperature compensated surface plasmon resonance (SPR) biosensor based on C-type special fiber is proposed and its application to DNA hybridization is demonstrated. Dual SPR effects were realized on the open microcavity by coating the C-type fiber with a gold film and filling the open microcavity with the temperature-sensitive material polydimethylsiloxane (PDMS). One effect occurs at the gold-analyte interface, where the presence of target DNA molecules interacting with the probe DNA immobilized on the fiber surface modulates the SPR response. The other effect occurs at the PDMS-gold interface, where temperature changes cause the change of PDMS refractive index (RI), altering the SPR response. The proposed sensor demonstrates exceptional capabilities. It can simultaneously detect DNA hybridization and temperature with high sensitivity. Specifically, the sensor exhibits a response of 0.11 nm/nM for DNA molecules and a significant response of −1.97 nm/°C for temperature. Additionally, it boasts a low limit of detection (LOD) of 7.9 nM, making it suitable for applications where early detection is crucial. Beyond its impressive performance, the sensor offers the advantages of compactness and ease of fabrication. This combination makes it a promising candidate for integration into portable devices, enabling on-site and real-time DNA detection with integrated temperature compensation. [ABSTRACT FROM AUTHOR]

With the development of technologies based on gold nanoparticles (AuNPs), bare AuNPs cannot meet the increasing requirements of biomedical applications. Modifications with different functional ligands are usually needed. DNA is not only the main genetic material, but also a good biological material, which has excellent biocompatibility, facile design, and accurate identification. DNA is a perfect ligand candidate for AuNPs, which can make up for the shortcoming of bare AuNPs. DNA-modified AuNPs (DNA-AuNPs) have exciting features and bright prospects in many fields, which have been intensively investigated in the past decade. In this review, we summarize the various approaches for the immobilization of DNA strands on the surface of AuNPs. Representative studies for biomedical applications based on DNA-AuNPs are also discussed. Finally, we present the challenges and future directions.

For anticancer therapy and disease prognosis in breast cancer, three PIK3CA point-mutations (H1047R, E545K, and E542K) play a significant role. To allow for specific and sensitive detection of these point-mutations with a hybridization-based detection concept, the assay conditions were optimized on a microarray technology platform. The resulting fluorescence-based microarray assay enables simultaneous and specific detection of three PIK3CA point-mutations. The optimized protocol was then adapted for use on a screen-printed and gold-plated silver sensor array with twelve working electrodes, one common counter electrode, and one common reference electrode. Chronoamperometric measurements employing an enzyme-amplified electrochemical assay allow for detecting PIK3CA point-mutations with a detection limit of 10 pM for short 24-mer target DNA. The mutant and the wild-type target DNA sequences gave significantly different signals in a broad concentration range of 1 nM – 100 nM, with the best separation found at 10 nM – 20 nM. Comparing the hybridization of short 24-mer and long 80-mer target DNA sequences reveals that the hybridization efficiency is reduced for long target DNA sequences. However, both the 24-mer and the 80-mer target DNA lead to successful detection of point-mutations. Finally, the electrochemical sensor allows for multiplexed detection of the three PIK3CA point-mutations.

Catch and release DNA decoys (CRDDs) utilize photochemically responsive nucleoside analogues that generate abasic sites upon exposure to light. Herein, we describe the synthesis and evaluation of four candidate CRDD monomers containing nucleobases that mimic endogenous pyrimidines: 2‐nitroimidazole (2‐NI), 2‐nitrobenzene (2‐NB), 2‐nitropyrrole (2‐NP) and 3‐nitropyrrole (3‐NP). Our studies reveal that 2‐NI and 2‐NP can function as CRDDs, whereas 3‐NP and 2‐NB undergo decomposition and transformation to a higher‐ordered structure upon photolysis, respectively. When incorporated into DNA, 2‐NP undergoes rapid photochemical cleavage of the anomeric bond (1.8 min half‐life) to yield an abasic site. Finally, we find that all four pyrimidine mimics show significantly greater stability when base‐paired against the previously reported 7‐nitroindole CRDD monomer. Our work marks the expansion of CRDD technology to both purine and pyrimidine scaffolds. [ABSTRACT FROM AUTHOR]

A highly sensitive silicon nanowire (SiNW)-based sensor device was developed using electron beam lithography integrated with complementary metal oxide semiconductor (CMOS) technology. The top-down fabrication approach enables the rapid fabrication of device miniaturization with uniform and strictly controlled geometric and surface properties. This study demonstrates that SiNW devices are well-aligned with different widths and numbers for pH sensing. The device consists of a single nanowire with 60 nm width, exhibiting an ideal pH responsivity (18.26 × 106 Ω/pH), with a good linear relation between the electrical response and a pH level range of 4–10. The optimized SiNW device is employed to detect specific single-stranded deoxyribonucleic acid (ssDNA) molecules. To use the sensing area, the sensor surface was chemically modified using (3-aminopropyl) triethoxysilane and glutaraldehyde, yielding covalently linked nanowire ssDNA adducts. Detection of hybridized DNA works by detecting the changes in the electrical current of the ssDNA-functionalized SiNW sensor, interacting with the targeted ssDNA in a label-free way. The developed biosensor shows selectivity for the complementary target ssDNA with linear detection ranging from 1.0 × 10−12 M to 1.0 × 10−7 M and an attained detection limit of 4.131 × 10−13 M. This indicates that the use of SiNW devices is a promising approach for the applications of ion detection and biomolecules sensing and could serve as a novel biosensor for future biomedical diagnosis. [ABSTRACT FROM AUTHOR]

A comprehensive investigation into how nanostructures alter real-time DNA hybridization kinetics in both buffer and complex media and under a wide range of probe and target concentrations is currently lacking. In response, we use a real-time, wash-free, and in situ assay to study DNA hybridization kinetics by performing continuous electrochemical measurements in different media. We investigated the differences in hybridization kinetics under three regimes of probe density (low, medium, and high) and over three orders of magnitude of target concentrations (0.01-1 μM). Additionally, we compared the performance of planar and nanostructured electrodes in buffer, blood, urine, and saliva. Our experiments indicate that adding nanostructures to the transducer surface is only effective under a specific probe/target concentration regime. Additionally, we found that direct electrochemical readout is possible in the examined physiological media, with measurements in blood showing the highest and saliva showing the lowest signal magnitudes compared to buffer.

Abstract Gold nanoparticles (Au NPs) based technology has been shown to possess enormous potential in the viral nucleic acid diagnosis. Despite significant advancement in this domain, the existing literature reveals the diversity in the conditions employed for hybridization and tagging of thiolated nucleic acid probes over the Au NPs. Here we employ the probe sequence derived from the Hepatitis C virus to identify the optimal hybridization and thiol-Au NP tagging conditions. In a typical polymerase chain reaction, the probes are initially subjected to flash heating at elevated temperatures to obtain efficient annealing. Motivated by this, in the current study, the hybridization between the target and the antisense oligonucleotide (ASO) has been studied at 65 °C with and without employing flash heating at temperatures from 75 to 95 °C. Besides, the efficiency of the thiolated ASO’s tagging over the Au NPs with and without citrate buffer has been explored. The study has revealed the beneficial role of flash heating at 95 °C for efficient hybridization and the presence of citrate buffer for rapid and effective thiol tagging over the Au NPs. The combinatorial effect of these conditions has been found to be advantageous in enhancing the sensitivity of ratiometric genosensing using Au NPs.

Milk is a widely consumed product, and its adulteration is not only common but also very dangerous. This study aimed to develop a biosensor for the detection of milk adulteration using DNA hybridization coupled with an electrochemical device. The advantages of biosensors over traditional laboratory methods, such as their speed, ease of use, and cost-effectiveness, are combined with the sensitivity of DNA hybridization. A capacitive biosensor was developed using interdigitated gold electrodes on paper substrate, which were modified with specific oligonucleotides for cow mitochondrial DNA that served as the biorecognition element. The methodology relies on the measurement of changes in capacitance due to DNA hybridization. Preliminary results are presented, showing the ability of this biosensor to detect bovine DNA in goat milk with high sensitivity and specificity. The results show that this biosensor has the potential to be a low-cost, easy-to-perform, and fast method for the detection of milk adulteration. This biosensor technology is a promising development for the detection of milk adulteration and can help to ensure the safety and quality of milk products.

A simple electrochemical DNA (E-DNA) biosensor for rapid and specific determination of DNA was developed based on electrical potential-assisted hybridization. The E-DNA biosensor was constructed by a capture probe immobilized on the gold electrode. An external alternating voltage was applied to the electrode interface during DNA hybridization between the capture probe with its target chain and a ferrocene-labeled peptide nucleic acid (Fc-PNA). The PNA was used to minimize the electrostatic repulsion between nucleic acids due to its neutral backbone. The hybridized DNA was examined by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopic (EIS). Compared with the passive hybridization strategy that generally takes several hours, this E-DNA biosensor took the advantage of controllable potential to accelerate nucleic acid hybridization and significantly shorten it to less than 90 s, thereby achieving rapid detection. The electrochemical signal showed a good linear relationship with the target DNA from 10 pM to 10 nM. In addition, this E-DNA biosensor had good specificity that was capable of distinguishing mismatched sequences. The developed biosensor is a promising diagnostic for the rapid and accurate determination of DNA. [ABSTRACT FROM AUTHOR]

In this article, we present a novel configuration of tin di-selenide (SnSe2) and zinc oxide (ZnO) based surface plasmon resonance (SPR) biosensor for DNA hybridization, urine abnormality, and anemia sensing. The configuration involves the stacked structure of prism (SF-10)/gold (Au)/ZnO/SnSe2/PBS. The performance parameters such as sensitivity (S), detection accuracy (DA), and figure of merit (FoM) are investigated at a fixed wavelength of 633 nm. Also, for SnSe2 the condition of zero chemical potential at room temperature was maintained. The obtained S, DA, and FoM are 153.3182 deg/RIU, 0.604 deg− 1and 9.15RIU− 1, respectively. [ABSTRACT FROM AUTHOR]

Panax ginseng and Panax quinquefolius, which are commonly called Chinese ginseng and American ginseng respectively, have different medicinal properties and market values; however, these samples can be difficult to differentiate from one another based on physical appearances of the samples especially when they are in powdery or granular forms. A molecular technique is thus needed to overcome this difficulty; this technique is based on the nucleic acid test (NAT) conducted on the microfluidic chip surface. Three single nucleotide polymorphism (SNP) sites (i.e. N1, N2, N3) on the Panax genome that differ between P. ginseng (G) and P. quinquefolius (Q) have been selected to design probes for the NAT. Primers were designed to amplify the antisense strands by asymmetric PCR. We have developed three different NAT methodologies involving surface immobilization and subsequent (stop flow or dynamic) hybridization of probes (i.e. N1G, N1Q, N2G, N2Q, N3Q) to the antisense strands. These NAT methods consist of two steps, namely immobilization and hybridization, and each method is distinguished by what is immobilized on the microfluidic chip surface in the first step (i.e. probe, target or capture strand). These three NATs developed are called probe-target method 1, target-probe method 2 and three-strand complex method 3. Out of the three methods, it was found that the capture strand-target-probe method 3 provided the best differentiation of the ginseng species, in which a 3' NH2 capture strand is first immobilized and the antisense PCR strand is then bound, while N2G and N3Q probes are used for detection of P. ginseng (G) and P. quinquefolius (Q) respectively. [ABSTRACT FROM AUTHOR]

With the globalization and complexity of the food supply chain, the market is becoming increasingly competitive and food fraudulent activities are intensifying. The current state of food detection faced two primary challenges. Firstly, existing testing methods were predominantly laboratory-based, requiring complex procedures and precision instruments. Secondly, there was a lack of accurate and efficient quantitative detection methods. Taking cow's milk as an example, this study introduced a novel method for nucleic acid quantification in dairy products, based on lateral flow strips (LFS). The core idea of this method is to design single-stranded DNA (ssDNA) probes to hybridize with mitochondrial genes, which are abundant, stable, and species-specific in dairy products, as detection targets. Drawing inspiration from the principles of nucleic acid amplification, this research innovatively established a new DNA hybridization method, named LAMP-Like Hybridization (HybLAMP-Like). Leveraging the denaturation and DNA polymerization functions of the bst enzyme, efficient binding of the probe and template strand was achieved. This method eliminated the need for nucleic acid amplification, simplifying the procedure and mitigating aerosol contamination, thereby ensuring the accuracy of the detection results. The method exhibited exceptional sensitivity, capable of detecting extremely low to 12.5 ng in visual inspection and 3.125 ng when using a reader. In terms of practicality, it could achieve visual detection of cow's milk content as low as 1% in adulterated dairy products. When combined with a portable LFS reader, it also enabled precise quantitative analysis of milk adulteration. [Display omitted] [ABSTRACT FROM AUTHOR]

The development of a simple and inexpensive DNA biosensor based on a gold nanoparticle (Au-NPs)/glutathione (GSH) matrix is reported to characterize DNA hybridization. The adhesion and morphological properties of the film were characterized using contact angle measurements and scanning electronic microscopy (SEM), respectively. The modified gold electrode was characterized by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The results indicate that DNA/AuNPs-GSH/cysteine provides excellent sensitivity for the target DNA in the linear range of response from 7 pM to 5 nM. The interaction of 6-thioguanine (6-tg) with the double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA) modified electrode was investigated using DPV and EIS. Consequently, 6-tg is accumulated onto the modified electrode causing a decrease of the charge transfer resistance (Rct) across the dynamic range from 1 pM to 10 µM. The developed electrochemical DNA biosensor was demonstrated to be efficient to characterize the interactions with drugs and micropollutants in wastewater. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) is an emerging human infectious disease caused by severe acute respiratory syndrome 2 (SARS-CoV-2, initially called novel coronavirus 2019-nCoV) virus. Thus, an accurate and specific diagnosis of COVID-19 is urgently needed for effective point-of-care detection and disease management. The reported promise of two-dimensional (2D) transition-metal carbides (Ti3C2Tx MXene) for biosensing owing to a very high surface area, high electrical conductivity, and hydrophilicity informed their selection for inclusion in functional electrodes for SARS-CoV-2 detection. Here, we demonstrate a new and facile functionalization strategy for Ti3C2Tx with probe DNA molecules through noncovalent adsorption, which eliminates expensive labeling steps and achieves sequence-specific recognition. The 2D Ti3C2Tx functionalized with complementary DNA probes shows a sensitive and selective detection of nucleocapsid (N) gene from SARS-CoV-2 through nucleic acid hybridization and chemoresistive transduction. The fabricated sensors are able to detect the SARS-CoV-2 N gene with sensitive and rapid response, a detection limit below 105 copies/mL in saliva, and high specificity when tested against SARS-CoV-1 and MERS. We hypothesize that the MXenes' interlayer spacing can serve as molecular sieving channels for hosting organic molecules and ions, which is a key advantage to their use in biomolecular sensing. [ABSTRACT FROM AUTHOR]

Nucleotide arrays require controlled surface densities and minimal nucleotide-substrate interactions to enable highly specific and efficient recognition by corresponding targets. We investigated chemical lift-off lithography with hydroxyl- and oligo(ethylene glycol)-terminated alkanethiol self-assembled monolayers as a means to produce substrates optimized for tethered DNA insertion into post-lift-off regions. Residual alkanethiols in the patterned regions after lift-off lithography enabled the formation of patterned DNA monolayers that favored hybridization with target DNA. Nucleotide densities were tunable by altering surface chemistries and alkanethiol ratios prior to lift-off. Lithography-induced conformational changes in oligo(ethylene glycol)-terminated monolayers hindered nucleotide insertion but could be used to advantage via mixed monolayers or double-lift-off lithography. Compared to thiolated DNA self-assembly alone or with alkanethiol backfilling, preparation of functional nucleotide arrays by chemical lift-off lithography enables superior hybridization efficiency and tunability.

In the past decade, a great effort has been devoted to develop new biosensor platforms for the detection of a wide range of analytes. Among the various approaches, magneto-DNA assay platforms have received extended interest for high sensitive and specific detection of targets with a simultaneous manipulation capacity. Here, using nitrogen-vacancy quantum centers in diamond as transducers for magnetic nanotags (MNTs), a hydrogel-based, multiplexed magneto-DNA assay is presented. Near-background-free sensing with diamond-based imaging combined with noninvasive control of chemically robust nanotags renders it a promising platform for applications in medical diagnostics, life science, and pharmaceutical drug research. To demonstrate its potential for practical applications, we employed the sensor platform in the sandwich DNA hybridization process and achieved a limit of detection in the attomolar range with single-base mismatch differentiation. [ABSTRACT FROM AUTHOR]

Biochemical reactions are typically slowed down by decreasing temperature. However, accelerated reaction kinetics have been observed for a long time. More recent examples have highlighted the unique role of freezing in fabricating supermaterials, degrading environmental contaminants, and accelerating bioreactions. Functional nucleic acids are DNA or RNA oligonucleotides with versatile properties, including target recognition, catalysis, and molecular co4mputing. In this review, we discuss the current observations and understanding of freezing-facilitated reactions involving functional nucleic acids. Molecular reactions such as ligation/conjugation, cleavage, and hybridization are discussed. Moreover, freezing-induced DNA-nanoparticle conjugations are introduced. Then, we describe our effect in immobilizing DNA on bulk surfaces. Finally, we address some critical questions and research opportunities in the field., (© 2024 Wiley-VCH GmbH.)

Gold nanoparticles (AuNPs) are popular labels for colorimetric detection of various analytes, involving proteins, nucleic acids, viruses, and whole cells because of their outstanding optical properties, inertness, and modification variability. In this work, we present an improved approach for enhancement of color intensity for DNA membrane microarrays based on seed-mediated growth of AuNP labels. Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microarrays. Then biotin is revealed by a streptavidin–AuNP conjugate followed by the detection of AuNPs. Optimization of seed-mediated enlargement of AuNPs by the reduction of tetrachloroauric acid with hydroxylamine made it possible to change the coloring of specific spots on the microarrays from pink to a more contrasting black with minor background staining. Mean size of the resulting AuNPs was four times larger than before the enhancement. Adjusting the pH of HAuCl4 solution to 3.5 and use of a large excess of hydroxylamine increased the signal/background ratio by several times. The method’s applicability was demonstrated for quantification of a short oligonucleotide of 19 bases and full-length TEM-type β-lactamase genes of 860 bp responsible for the development of bacterial resistance against β-lactam antibiotics. Improved protocol for AuNP enlargement may be further transferred to any other membrane-based assays of nucleic acids with both instrumental and visual colorimetric detection.

Quartz-crystal microbalance (QCM) is a technique that can measure nanogram-order masses. When a receptor is immobilized on the sensor surface of a QCM device, the device can detect chemical molecules captured by the mass change. Although QCM devices have been applied to biosensors that detect biomolecules without labels for biomolecular interaction analysis, most highly sensitive QCM devices are benchtop devices. We considered the fabrication of an IC card-sized QCM device that is both portable and battery-powered. Its miniaturization was achieved by repurposing electronic components and film batteries from smartphones and wearable devices. To demonstrate the applicability of the card-sized QCM device as a biosensor, DNA-detection experiments were performed. The card-sized QCM device could detect specific 10-mer DNA chains while discerning single-base differences with a sensitivity similar to that of a conventional benchtop device. The card-sized QCM device can be used in laboratories and in various other fields as a mass sensor.

Gold nanoparticles (Au NPs) based technology has been shown to possess enormous potential in the viral nucleic acid diagnosis. Despite significant advancement in this domain, the existing literature reveals the diversity in the conditions employed for hybridization and tagging of thiolated nucleic acid probes over the Au NPs. Here we employ the probe sequence derived from the Hepatitis C virus to identify the optimal hybridization and thiol-Au NP tagging conditions. In a typical polymerase chain reaction, the probes are initially subjected to flash heating at elevated temperatures to obtain efficient annealing. Motivated by this, in the current study, the hybridization between the target and the antisense oligonucleotide (ASO) has been studied at 65 °C with and without employing flash heating at temperatures from 75 to 95 °C. Besides, the efficiency of the thiolated ASO's tagging over the Au NPs with and without citrate buffer has been explored. The study has revealed the beneficial role of flash heating at 95 °C for efficient hybridization and the presence of citrate buffer for rapid and effective thiol tagging over the Au NPs. The combinatorial effect of these conditions has been found to be advantageous in enhancing the sensitivity of ratiometric genosensing using Au NPs. [ABSTRACT FROM AUTHOR]

As detection of specific deoxyribonucleic acid (DNA) sequences is of great importance in diagnosing many fatal and genetic diseases, there have been renewed interests in designing highly sensitive label-free DNA detection platforms. This paper presents a molybdenum diselenide (MoSe2)-graphene hybrid structure-based surface plasmon resonance (SPR) biosensor with capability of detecting single-nucleotide polymorphism (SNP) and DNA hybridization events. Because of its better absorption capability the MoSe2 layer provides strong excitation energy which induces huge charge carrier transfer while the graphene layer serves as a biomolecular recognition element to capture bio-analytes through π-stacking force. The silver (Ag) layer is attached to the BK7 prism by a very thin titanium (Ti) adhesion film which also serves as oxidization and corrosion protection layer. The biosensor is optimized by numerical modeling and the optimum structure has an excellent sensitivity of 215.5°/RIU which is better than other reported works. It has been demonstrated that when target DNAs are attached to the probe DNAs pre-immobilized on the sensing surface the change of SPR angle is insignificant for mismatched DNA strands while it is highly prominent for complementary DNA strands. Thus, the proposed sensor can effectively distinguish hybridization event and SNP by examining the level of changes in resonance angle and reflectance spectrum. [ABSTRACT FROM AUTHOR]

Field-effect transistors (FET) using low-dimensional materials has attracted attention for detection of DNA hybridization. Here, we proposed a photocurrent-modulated FET sensor, where the I p h o t o c u r r e n t (g a t e) + I p h o t o c u r r e n t (D N A) dramatically enhance the response signal, I c u r r e n t , compared to a traditional FET sensor. The proposed sensor successfully detects DNA signals with a limit of detection (LOD) down to attomolar concentrations that is nearly 100x lower than a traditional FET sensor. The sensing mechanism is postulated and discussed. The results suggest that photo-coupled modulation opens new avenues for further development of the FET in biosensing applications. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Emerging antimicrobial drug resistance is increasing the complexity involved in treating critical conditions such as bacterial induced sepsis. Methods for diagnosing specific drug resistance tend to be rapid or sensitive, but not both. Detection methods like sequence-specific single-molecule analysis could address this concern if they could be adapted to work on smaller targets similar to those produced in traditional clinical situations. In this work we demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be created by immobilizing DNA with a biotin/streptavidin magnetic bead system, labeling with SYBR Gold, and rinsing the excess away while the probe retains multiple fluorophores. These probes with multiple fluorophores can then be used to label a bacterial plasmid target in a sequence-specific manner. These probes enabled the detection of 1 pM plasmid samples containing a portion of an antibiotic resistance gene sequence. This system shows the possibility of improving capture and fluorescence labeling of small nucleic acid fragments, generating lower limits of detection for clinically relevant samples while maintaining rapid processing times.

A highly sensitive silicon nanowire (SiNW)-based sensor device was developed using electron beam lithography integrated with complementary metal oxide semiconductor (CMOS) technology. The top-down fabrication approach enables the rapid fabrication of device miniaturization with uniform and strictly controlled geometric and surface properties. This study demonstrates that SiNW devices are well-aligned with different widths and numbers for pH sensing. The device consists of a single nanowire with 60 nm width, exhibiting an ideal pH responsivity (18.26 × 106 Ω/pH), with a good linear relation between the electrical response and a pH level range of 4–10. The optimized SiNW device is employed to detect specific single-stranded deoxyribonucleic acid (ssDNA) molecules. To use the sensing area, the sensor surface was chemically modified using (3-aminopropyl) triethoxysilane and glutaraldehyde, yielding covalently linked nanowire ssDNA adducts. Detection of hybridized DNA works by detecting the changes in the electrical current of the ssDNA-functionalized SiNW sensor, interacting with the targeted ssDNA in a label-free way. The developed biosensor shows selectivity for the complementary target ssDNA with linear detection ranging from 1.0 × 10−12 M to 1.0 × 10−7 M and an attained detection limit of 4.131 × 10−13 M. This indicates that the use of SiNW devices is a promising approach for the applications of ion detection and biomolecules sensing and could serve as a novel biosensor for future biomedical diagnosis.