Thiol-modified Oligonucleotides Research Articles

Codeposition Enhances the Performance of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors, a minimally invasive means of performing high-frequency, real-time measurement of drugs and biomarkers in situ in the body, have traditionally been fabricated by depositing their target-recognizing aptamer onto an interrogating gold electrode using a "sequential" two-step method involving deposition of the thiol-modified oligonucleotide (typically for 1 h) followed by incubation in mercaptohexanol solution (typically overnight) to complete the formation of a stable, self-assembled monolayer. Here we use EAB sensors targeting vancomycin, tryptophan, and phenylalanine to show that "codeposition", a less commonly employed EAB fabrication method in which the thiol-modified aptamer and the mercaptohexanol diluent are deposited on the electrode simultaneously and for as little as 1 h, improves the signal gain (relative change in signal upon the addition of high concentrations of the target) of the vancomycin and tryptophan sensors without significantly reducing their stability. In contrast, the gain of the phenylalanine sensor is effectively identical irrespective of the fabrication approach employed. This sensor, however, appears to employ binding-induced displacement of the redox reporter rather than binding-induced folding as its signal transduction mechanism, suggesting in turn a mechanism for the improvement observed for the other two sensors. Codeposition thus not only provides a more convenient means of fabricating EAB sensors but also can improve their performance.

Disposable microRNA biosensors based on dual-role polymer-dispersed silver nanowires for diagnosis of viral hemorrhagic septicemia virus infection in olive flounder

Fish farms often suffer from viral infections that can lead to mass mortality. Early detection of these infections can help prevent the spread of the infection. microRNA (miRNA) is a new biomarker for detecting early infections in farmed olive flounder. We developed a disposable miRNA biosensor by utilizing screen-printed carbon electrodes with polymer-dispersed silver nanowires (P-AgNWs). The target miRNA here was miR-15b-5p, which is considered a specific candidate for detecting viral hemorrhagic septicemia virus (VHSV) infection in fish. A capture probe (CP), a thiol-modified DNA oligonucleotide chain complementary to the miR-15b-5p, was immobilized on the surface of P-AgNWs. While the hybridization of miR-15b-5p with the CP is monitored using square wave voltammetry techniques based on the specific binding of methylene blue (MB) to the DNA-RNA hybrid formed between CP and the miR-15b-5p. The miRNA biosensor demonstrated a wide linear response range of 1.0–100 fM under optimum conditions with a low detection limit of 0.70 fM. According to a series of voltammetric experiments, the approach demonstrated acceptable precision and sensitivity for determining the miR-15b-5p levels in olive flounder infected with VHSV.

Modified gold nanoparticle colorimetric probe-based biosensor for direct and rapid detection of Mycobacterium tuberculosis in sputum specimens

The incidence of Mycobacterium tuberculosis (MTB) is increasing due to lack of appropriate diagnostic and therapeutic methods. Therefore, early and accurate detection of this bacteria plays a significant role in controlling tuberculosis. This study aimed to design, develop, and implement a direct and rapid detection method of MTB using modified gold nanoparticle (AuNP) colorimetric probe-based biosensor in sputum specimens. Spherical AuNPs were synthesized by the citrate reduction method and were functionalized using thiol-modified oligonucleotides (AuNP-biosensor). AuNP-biosensor and IS6110 PCR were compared to the gold standard in terms of analytical and clinical sensitivity and specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy in 52 clinical specimens. Gold standard was defined as a positive result in concentrated sputum smear microscopy (SSM), culture, or Xpert MTB/RIF.The AuNP-biosensor had 100% sensitivity and specificity for detection of total sputum DNA in less than 15min with ready-to-use AuNP-biosensor. PPV, NPV, DOR and accuracy of this method were 100%, 100%, 2325 and 100%, respectively. Considering the promising results of the diagnostic value indices of the AuNP-biosensor, the designed method is an affordable, rapid, reliable, and cost-beneficial way for direct detection of MTB in sputum specimens.

Development of aptamer based colorimetric assay for whole blood glycated hemoglobin (HbA1c) estimation using gold nanoparticles

Thiol-modified aptamer oligonucleotides containing gold nanoparticles (G@NPs) bound to glycated hemoglobin (HbA1c) with high affinity in whole blood samples. Aptamer thiol groups enhanced the stability of aptamers adsorbed on the surface of G@NPs. A detection strategy was used to visually confirm the presence of HbA1c and the absorbance of the colored compounds at selected wavelengths. Modified G@NPs are used in many colorimetric detection strategies due to their high extinction coefficient and strongly distance-dependent optical properties. In the present study, as the target analyte reports a method to prepare gold nanoparticle-bound purple aptamer aggregates that rapidly collapse into bluish red dispersed nanoparticles upon binding of HbA1c. HbA1c was detected in unpretreated whole blood after dilution at pH 7.4 and with UV-VIS double beam spectrophotometer with a lower limit of detection (LOD) (0.1 μM) and a much broader linear detection range (0.1 μM–100 μM). This current colorimetric method provides a rapid (7 min), accurate and high determination of whole blood HbA1c with minimal interference from uric acid, bovine serum albumin, hemoglobin, and glucose. The lower detection limit and broader linearity of aptamer-bound G@NPs hybrids improve the accuracy and precision of HbA1c measurements.

A "turn-on" fluorescence resonance energy transfer aptasensor based on carbon dots and gold nanoparticles for 17β-estradiol detection in sea salt.

17β-estradiol is abused in the food industry. Excess 17β-estradiol can disturb the endocrine system or cause many diseases including obesity, diabetes, cardiac-cerebral vascular disease, and cancers in the human body. A "turn-on" fluorescence resonance energy transfer (FRET) aptasensor based on carbon dots (CDs) and gold nanoparticles (AuNPs) was developed for the detection of 17β-estradiol. A thiol-modified oligonucleotide was conjugated to AuNPs and amino modified oligonucleotide was linked to CDs. The 17β-estradiol aptamer was hybridized with the two oligonucleotides, shortening the distance between CDs and AuNPs. With 360 nm UV light excitation, FRET occurred between CDs and AuNPs. The system was "turn-off". When 17β-estradiol was detected, the aptamer specifically bound to 17β-estradiol, and the FRET system was destroyed, leading to the "turn-on" phenomenon. The fluorescence intensity recovery was detected in the concentration range of 400 pM to 5.5 μM. The limit of detection (LOD) was 245 pM. The FRET aptasensor demonstrated good selectivity for 17β-estradiol detection. Reasonable spiked recoveries were obtained in sea salt samples. It showed the potential for estrogen detection in food safety and environmental applications.

Modified gold nanoparticle colorimetric probe-based biosensor coupled with allele-specific PCR for rapid detection of G944C mutation associated with isoniazid resistance

The emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) makes tuberculosis (TB) difficult to control and treat. Therefore, rapid and reliable detection of MDR-TB within the community plays a significant role. The aim of this study was to investigate the use of modified gold nanoparticle (AuNP) colorimetric probe-based biosensor coupled with allele-specific PCR for the rapid detection of G944C mutation associated with isoniazid resistance in MTB. Based on the citrate reduction method, the AuNPs were synthesized and functionalized using thiol-modified oligonucleotides (AuNP-biosensor). The AuNP-biosensor method was compared to the gold standard in terms of analytical and clinical sensitivity and specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy. The gold standard was defined as a positive result in sequencing.The AuNP-biosensor had 100% sensitivity and specificity for detection of G944C mutation. After allele-specific PCR amplification the results were carried out for <15 min with ready-to-use AuNP-biosensor. PPV, NPV, DOR and accuracy of this method were 100%, 100%, 309 and 100%, respectively. The method designed in this study can identify the desired mutation with high efficiency after initial amplification, either from the sputum sample or from isolated bacteria. By designing a panel of this method, different MTB resistance-associated mutations could be detected more rapidly.

DNA Nanostructure-Guided Plasmon Coupling Architectures

DNA Nanostructure-Guided Plasmon Coupling Architectures

A novel electrochemical biosensor for detection of micrococcal nuclease in milk based on a U-shaped DNA structure

Micrococcal nuclease (MNase) is an extracellular endo-exonuclease of Staphylococcus aureus (S. aureus) that can hydrolyze single and double-stranded DNA and RNA. The presence of MNase is used as an outstanding diagnostic biomarker for the identification of S. aureus. Herein, we designed a novel U-shaped electrochemical biosensor to sensitively detect MNase using a gold electrode, thiol-modified oligonucleotide strands and their complementary strand (CS). The principle of the sensing platform is that in the absence of MNase, a U-shaped DNA structure on the gold electrode surface prohibits [Fe(CN)6]3-/4- from reaching the electrode surface through physical inhibition and electrostatic repulsion. Following the addition of MNase, the oligonucleotide sequences are digested into the short DNA sections, which leads the redox agent access to the surface of the working electrode and a sharp current peak. This analytical method can detect MNase in a linear range of 0.00022–0.02 U/μL with the detection limit of 2.37 × 10−5 U/μL. The presented biosensor showed satisfying results for the MNase recognition in the spiked milk samples. Moreover, the biosensor exhibited a prominent electrochemical signal when exposed to MNase secreted from S. aureus in the culture medium sample. Overall, the proposed biosensor has a strong potential for highly selective and sensitive detection of MNase.

Probing DNA Stability in High Electric Fields: Electrochemical Melting Analysis

Here we present our results on the electric field-induced melting of surface-bound DNA (i.e. electrochemical melting). In particular, we examine the effects of the DNA-binding compound cisplatin on the stability of DNA within high electric fields. Cisplatin is an anticancer drug that has been used to successfully treat testicular, ovarian, and bladder cancers, among others (1). It crosslinks DNA between the N7 atoms of purine bases, resulting in changes in structure and stability, ultimately interfering with vital cellular processes in vivo.Self-assembled monolayers (SAMs) of thiol-modified oligonucleotides on gold electrodes provide selective and sensitive interfaces for the development of chemical and biological sensors (2). While many transduction mechanisms have been utilized (including electrochemical, optical, and gravimetric) and many analytes targeted (e.g. small DNA-binding molecules, heavy metals, and specific DNA sequences), the unique steric and electrostatic environment of the crowded DNA in the electrical double-layer complicates the behavior of these biosensors (3). Electrical potentials applied at the DNA-SAM have been shown to modulate the behavior of these monolayers (4). Here, we utilize the electric field-effect to induce melting of the bound doublestranded DNA, and we monitor the kinetics of the melting process using square wave voltammetry (5).We find that the effect of cisplatin on the melting behavior depends on the method used to the prepare the monolayer, in particular the melting depends on surface-coverage and heterogeneity of the resulting layers. Under some conditions, cisplatin results in an apparent stabilization of the DNA-SAM, and a destabilization in others. Significant differences in the resulting melting curves suggest that the mode of cisplatin-DNA binding varies depending on the SAM preparation method. The methodology presented here has the potential of (1) providing an electrochemical approach for studying the interaction between DNA and small molecules, and (2) providing a method for indirectly assessing the heterogeneity of DNA-SAMs via electrochemical melting in the presence of cross-linking agents.1. R. A. Alderden, M. D. Hall and T. W. Hambley, Journal of chemical education, 83, 728 (2006).2. J. J. Gooding and N. Darwish, The Chemical Record, 12, 92 (2012).3. P. Gong and R. Levicky, Proceedings of the National Academy of Sciences, 105, 5301 (2008).4. U. Rant, K. Arinaga, S. Fujita, N. Yokoyama, G. Abstreiter and M. Tornow, Organic & biomolecular chemistry, 4, 3448 (2006).5. D. Ho, W. Hetrick, N. Le, A. Chin and R. M. West, Journal of The Electrochemical Society, 166, B236 (2019).

Response of HPRT Gene Fragment Functionalized Gold Nanoparticles to Gamma Ray Irradiation.

Radiation-sensitive biomolecules are highly significant for studying biological effects of radiation and developing ionizing radiation detectors based on biomolecules. In this work, we selected hypoxanthine phosphoribosyl transferase gene fragments sensitive to gamma-ray irradiation as a sensing element for radiation detection. The end was modified with thiol groups. The thiol-modified oligonucleotide sequences were coupled to the surface of gold nanoparticles by Au-S covalent bonds. The DNA attached to the surface of gold nanoparticles forms a DNA-AuNPs assembly through base pairing. The assembly was irradiated by gamma rays. And its response to radiation was studied with ultraviolet-visible spectroscopy and surface-enhanced Raman scattering (SERS) spectroscopy techniques. SERS spectroscopy and ultraviolet spectroscopy can detect the response of the DNA-AuNPs assembly to gamma-ray irradiation below 100 and 100 - 250 Gy, respectively. The results indicated that it was feasible to develop a new approach of gamma-ray detectors using biomolecular assemblies of gold nanoparticles.

Facet-selective asymmetric functionalization of anisotropic gold nanoprisms for Janus particle synthesis

Janus particles, which possess asymmetric structures and/or multiple surface chemistries, have attracted intense interest due to their capability of multiple distinct interactions with their environment. Most Janus particles synthesis techniques rely on applying coating molecules on isotropic particles. Herein, we demonstrate a method to controllably functionalize both major facets of geometrically anisotropic triangular gold nanoprisms in the size range of 100–200 nm with two distinct molecular coatings (hexadecane thiol or thiolated poly (ethylene) glycol and thiol-modified oligonucleotide), giving rise to the formation of Janus gold nanoprisms. Janus particles orient themselves on the interface of water-chloroform mixture and exhibit variable interaction with both hydrophilic and hydrophobic surfaces due to their amphiphilicity and undergo asymmetric self-assembly behavior with other nanoparticles.

Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles.

The current outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) demands its rapid, convenient, and large-scale diagnosis to downregulate its spread within as well as across the communities. But the reliability, reproducibility, and selectivity of majority of such diagnostic tests fail when they are tested either to a viral load at its early representation or to a viral gene mutated during its current spread. In this regard, a selective “naked-eye” detection of SARS-CoV-2 is highly desirable, which can be tested without accessing any advanced instrumental techniques. We herein report the development of a colorimetric assay based on gold nanoparticles (AuNPs), when capped with suitably designed thiol-modified antisense oligonucleotides (ASOs) specific for N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2, could be used for diagnosing positive COVID-19 cases within 10 min from the isolated RNA samples. The thiol-modified ASO-capped AuNPs agglomerate selectively in the presence of its target RNA sequence of SARS-CoV-2 and demonstrate a change in its surface plasmon resonance. Further, the addition of RNaseH cleaves the RNA strand from the RNA–DNA hybrid leading to a visually detectable precipitate from the solution mediated by the additional agglomeration among the AuNPs. The selectivity of the assay has been monitored in the presence of MERS-CoV viral RNA with a limit of detection of 0.18 ng/μL of RNA having SARS-CoV-2 viral load. Thus, the current study reports a selective and visual “naked-eye” detection of COVID-19 causative virus, SARS-CoV-2, without the requirement of any sophisticated instrumental techniques.

Probing DNA Stability in High Electric Fields: Electrochemical Melting Analysis

Here we present our results on the electric field-induced melting of surface-bound DNA (i.e. electrochemical melting). In particular, we examine the effects of DNA-binding compounds, such as cisplatin, on the stability of DNA within high electric fields. Cisplatin is an anticancer drug that has been used to successfully treat testicular, ovarian, and bladder cancers, among others [1]. It crosslinks DNA between the N7 atoms of purine bases, resulting in changes in structure and stability, ultimately interfering with vital cellular processes in vivo. Self-assembled monolayers (SAMs) of thiol-modified oligonucleotides on gold electrodes provide selective and sensitive interfaces for the development of chemical and biological sensors [2]. While many transduction mechanisms have been utilized (including electrochemical, optical, and gravimetric) and many analytes targeted (e.g. small DNA-binding molecules, heavy metals, and specific DNA sequences), the unique steric and electrostatic environment of the crowded DNA in the electrical double-layer complicates the behavior of these biosensors [3]. Electrical potentials applied at the DNA-SAM have been shown to modulate the behavior of these monolayers [4]. Here, we utilize the electric field-effect to induce melting of the bound double-stranded DNA, and we monitor the kinetics of the melting process using square wave voltammetry [5]. We find that the effect of cisplatin on the melting behavior depends on the method used to the prepare the monolayer, in particular the surface-coverage and heterogeneity of the resulting layers. In some instances, cisplatin results in a stabilization of the DNA-SAM and in others a destabilization. Significant differences in the resulting melting curves imply that the mode of cisplatin-DNA binding varies depending on the preparation method. This methodology has the potential of (1) providing an electrochemical approach for studying the interaction between DNA and small molecules, and (2) providing a method for indirectly assessing the heterogeneity of DNA-SAMs via electrochemical melting in the presence of cross-linking agents.[1] R. A. Alderden, M. D. Hall, T. W. Hambley, The discovery and development of cisplatin, Journal of chemical education, 83(5), (2006) 728.[2] J. J. Gooding and N. Darwish, The rise of self‐assembled monolayers for fabricating electrochemical biosensors—an interfacial perspective, The Chemical Record, 12, (2012) 92.[3] Gong and R. Levicky, DNA surface hybridization regimes, Proceedings of the National Academy of Sciences, 105, (2008) 5301.[4] U. Rant, K. Arinaga, S. Fujita, N. Yokoyama, G. Abstreiter, M. Tornow, Electrical manipulation of oligonucleotides grafted to charged surfaces, Organic & Biomolecular Chemistry 4 (2006) 3448.[5] D. Ho, W. Hetrick, N. Le, A. Chin, R. M. West, Electric Field Induced DNA Melting with Detection by Square Wave Voltammetry, Journal of the Electrochemical Society, 166, (2019) B236.

Synthesis of Small Gold Nanorods and Their Subsequent Functionalization with Hairpin Single Stranded DNA.

Small gold nanorods have a significantly large absorption/scattering ratio and are especially beneficial in exploiting photothermal effects, for example in photothermal therapy and remote drug release. This work systematically investigates the influence of growth conditions on the size, growth yield, and stability of small gold nanorods. The silver-assisted seed-mediated growth method was optimized to synthesize stable small gold nanorods with a high growth yield (>85%). Further study on the influence of silver ions on the growth facilitates the growth of small gold nanorods with tunable longitudinal surface plasmon resonance from 613 to 912 nm, with average dimensions of 13–25 nm in length and 5–6 nm in diameter. Moreover, the small gold nanorods were successfully functionalized with thiol-modified hairpin oligonucleotides (hpDNA) labeled with Cy5. Fluorescence intensity measurements show an increase in the presence of target DNA and an enhanced signal/background ratio when the longitudinal surface plasmon resonance of small gold nanorods overlaps with the excitation and emission wavelength of Cy5. This coincides with a reduced fluorescence lifetime of Cy5 in the hairpin structure, indicating surface plasmon resonance-enhanced energy transfer to the small gold nanorods. This study may provide insight on the synthesis and functionalization of small gold nanorods in biomedical sensing and therapy.

An Electrochemiluminescence Sensor Based on CdSe@CdS and God Signal Amplification for Sensitive Detection of Hg2+

Mercury is highly toxic, so it is important to develop a rapid and sensitive method for the detection of mercury. In this paper, mercaptopropionic acid-capped core-shell CdSe@CdS quantum dots (QDs) were synthesized by one-step hydrothermal method[1]. Molybdenum disulfide (MoS2) nanosheets were prepared by the method which we previously reported[2]. 5'-amino-modified oligonucleotide capture probe DNA1 (5'-NH2-ss-DNA) and 5'-thiol-modified oligonucleotide signal probe DNA2 (5'-SH-ss-DNA) was synthesized by Shanghai Bioengineering Technology Co., Ltd. (China). The sequence (5' to 3') is as follows[3]: DNA1：5'-NH2-AAAATTTTGCTTTGGTTT-3' DNA2：5'-SH-AAAAATTTCCTTTGCTTTT-3' Polycationic poly(diallyldimethylammonium chloride) (PDDA) and negatively charged QDs were adsorbed on the surface of MoS2, in sequence, to form the QDs- PDDA-MoS2 composites, which were employed as a matrix for the immobilization of DNA1 strand[1]. Gold nanoparticles (AuNPs) were prepared and were combined with the 5'-thiol-modified DNA2 probe and glucose oxidase (GOD) to prepare GOD-AuNPs-DNA2 conjugates[2]. First, the QDs-PDDA-MoS2 nanocomposites were modified onto the surface of a glassy carbon electrode (GCE) as a carrier for preparing an electrochemiluminescence (ECL) sensor. The QDs-PDDA-MoS2/GCE electrode was immersed in an aqueous solution consists of 10.0 mM ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and 5.0 mM N-hydroxysuccinimide (NHS) for 30 min to activate the terminal carboxylic acid group of QDs. DNA1 could immobilize onto the surface of the CdSe@CdS QDs via the interaction between the activated carboxylic acid group and the amino group of DNA1. The modified electrode was denoted as DNA1/QDs-PDDA-MoS2/GCE. The schematic diagram of fabrication of the sensor was shown in Fig. 1. Fig. 1 Frabrication process and the detection machanisms of the ECL sensor. Insert: curve (a) absence and (b) presence of Hg2+. Electrochemical measurement for ECL was carried out on an MPI-A detection system (Xi'an Rimax Electronics Co., Ltd. China) with a three-electrode system and a photomultiplier tube. The voltage was set at 800 V. A glassy carbon electrode (diameter, 3 mm) was used as a working electrode; a platinum wire and an Ag/AgCl (saturated KCl solution) were used as a counter electrode and a reference electrode, respectively. The DNA1/QDs-PDDA-MoS2/GCE electrodes were incubated with GOD-AuNPs-DNA2 suspension and different concentrations of Hg2+ solution in turns. Then the electrodes were immersed in 0.1 M phosphate buffer solution containing 1% glucose to measure ECL signal, respectively. The potential window was from 0 to 1.5 V with a scan rate of 100 mV/ s. Hg2+ ions can specifically bind to the T-T mismatch base of DNA1 and DNA2 to form the T-Hg2+-T complex. Therefore, GOD-AuNPs-DNA2 can be modified to the electrode surface. GOD can catalyze the oxidation reaction of dissolved oxygen molecules towards glucose in the solution to produce H2O2 which is a co-reactant and can enhance the ECL of the QDs. The ECL signal increased linearly with Hg2+ concentration from 1.0×10-12 to 1.0×10-6 M. The detection limit of 0.1 pM can be estimated at a signal-to-noise ratio of 3. The ECL sensor has the advantages of simple preparation method, high sensitivity, good specificity and wide linear range for the determination of Hg2+ ions.

Immobilization of gold nanoparticles with rhodamine to enhance the fluorescence resonance energy transfer between quantum dots and rhodamine; new method for downstream sensing of infectious bursal disease virus.

Infectious bursal disease virus is a causative agent of one of the most important disease which causes frequent tragic disaster in the poultry industry all over the world. Therefore, in the present study a new fluorescence resonance energy transfer-based technique was developed to detect VP2 gene of infectious bursal disease virus using two oligonucleotide probes labeled with quantum dots and rhodamine- immobilized gold nanoparticles (AuNPs-Rh). Quantum dots labeled with an amino-modified first oligonucleotide, and AuNPs-Rh labeled with thiol-modified second oligonucleotides were added to the DNA targets upon which hybridization occurred. In the presence of target the AuNPs-Rh will be located in the vicinity of the quantum dots and leads to the fluorescence resonance energy transfer to be occurred and subsequently the fluorescence intensity of quantum dots was stimulated. The immobilization of rhodamine to the surface of AuNPs increased the fluorescence intensity of rhodamine. The maximum fluorescence resonance energy transfer efficiency for the developed sensor is monitored at a quantum dots-PA/AuNPs-Rh-PT molar ratio of 1:10. Moreover, the feasibility of the developed nanobiosensor was demonstrated by the detection of a synthetic 49-mer nucleotide derived from infectious bursal disease virus and the limit of detection was estimated as 3 × 10-8 M. The developed DNA detection scheme is a simple, rapid and efficient technique which does not need excessive washing and separation steps.

A Markedly Improved Synthetic Approach for the Preparation of Multifunctional Au-DNA Nanoparticle Conjugates Modified with Optical and MR Imaging Probes.

We describe a new, and vastly superior approach for labeling spherical nucleic acid conjugates (SNAs) with diagnostic probes. SNAs have been shown to provide the unique ability to traverse the cell membrane and deliver surface conjugated DNA into cells while preserving the DNA from nuclease degradation. Our previous work on preparing diagnostically labeled SNAs was labor intensive, relatively low yielding, and costly. Here, we describe a straightforward and facile preparation for labeling SNAs with optical and MR imaging probes with significantly improved physical properties. The synthesis of Gd(III) labeled DNA Au nanoparticle conjugates is achieved by sequential conjugation of 3'-thiol-modified oligonucleotides and cofunctionalization of the particle surface with the subsequent addition of 1,2 diothiolate modified chelates of Gd(III) (abbreviated: DNA-GdIII@AuNP). This new generation of SNA conjugates has a 2-fold increase of DNA labeling and a 1.4-fold increase in Gd(III) loading compared to published constructs. Furthermore, the relaxivity ( r1) is observed to increase 4.5-fold compared to the molecular dithiolane-Gd(III) complex, and 1.4-fold increase relative to previous particle constructs where the Gd(III) complexes were conjugated to the oligonucleotides rather than directly to the Au particle. Importantly, this simplified approach (2 steps) exploits the advantages of previous Gd(III) labeled SNA platforms; however, this new approach is scalable and eliminates modification of DNA for attaching the contrast agent, and the particles exhibit improved cell labeling.

A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor

To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([CuII(phen)2]2+), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [CuII(phen)2]2+, a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [CuII(phen)2]2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [CuI(phen)2]+, with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [CuII(phen)2]2+.

Electric Field Induced Melting: Effect of Non-Specifically Absorbed DNA

Electric-field induced melting of surface-bound DNA can be carried out using electrochemical detection without parallel spectroscopic monitoring. With proper selection of applied potentials, ionic strength, and DNA coverage, discrimination of single mismatches based on the kinetics of DNA melting can be carried out quickly and reproducibly. Electrodes are prepared by the sequential exposure to thiol-modified oligonucleotides, mercaptohexanol, and finally hybridization with complimentary target oligonucleotides. The target is subsequently melted off the electrode by applying a sufficiently negative potential. The mechanism of e-melting is up for debate but likely involves the electrostatic repulsion between DNA’s phosphate backbone and the electrode surface, destabilization of the base pair hydrogen bonding or pi stacking, or some combination of these effects and others. The mercaptohexanol passivation layer plays an important role for electrochemical DNA sensing, particularly when the effects of electric field are crucial to the detection scheme as is the case in electrochemical melting. The mercaptohexanol layer, or some other alkyl thiol layer is almost universally used to remove non-specifically absorbed DNA from the surface and prevent further non-specific absorption. Physisorbed oligonucleotides that are not removed by the passivation layer are expected to have ill-defined interaction energies with the surface and will be expelled from the surface at potentials and rates different than the hybridized target oligonucleotides. Additionally, a uniform mercaptohexanol layer is necessary for a uniform electric field. In this work, we examine the extent to which non-specific absorption occurs even when the mercaptohexanol layer is present. We examine how non-specific absorption effects the melting behavior, including the melting potential, melting rate, and the reproducibility. Finally, we compare various methods for mitigating non-specific absorption. These methods include the use of surfactants during electrode modification, applications of potential pulse sequences to destabilize and remove physisorbed oligomers, and the use of elevated temperatures.

Gold nanoparticle-based probes for the colorimetric detection of Mycobacterium avium subspecies paratuberculosis DNA

Gold nanoparticle (AuNP) is considered to be the most stable metal nanoparticle having the ability to be functionalized with biomolecules. Recently, AuNP-based DNA detection methods captured the interest of researchers worldwide. Paratuberculosis or Johne's disease, a chronic gastroenteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), was found to have negative effect in the livestock industry. In this study, AuNP-based probes were evaluated for the specific and sensitive detection of MAP DNA. AuNP-based probe was produced by functionalization of AuNPs with thiol-modified oligonucleotide and was confirmed by Fourier-Transform Infrared (FTIR) spectroscopy. UV–Vis spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize AuNPs. DNA detection was done by hybridization of 10 μL of DNA with 5 μL of probe at 63 °C for 10 min and addition of 3 μL salt solution. The method was specific to MAP with detection limit of 103 ng. UV–Vis and SEM showed dispersion and aggregation of the AuNPs for the positive and negative results, respectively, with no observed particle growth. This study therefore reports an AuNP-based probes which can be used for the specific and sensitive detection of MAP DNA.