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Biosensors & Bioelectronics Mar 2021An electrical immuno-sandwich assay utilizing an electrokinetic-based streaming current method for signal transduction is proposed. The method records the changes in...
An electrical immuno-sandwich assay utilizing an electrokinetic-based streaming current method for signal transduction is proposed. The method records the changes in streaming current, first when a target molecule binds to the capture probes immobilized on the inner surface of a silica micro-capillary, and then when the detection probes interact with the bound target molecules on the surface. The difference in signals in these two steps constitute the response of the assay, which offers better target selectivity and a linear concentration dependent response for a target concentration within the range 0.2-100 nM. The proof of concept is demonstrated by detecting different concentrations of Immunoglobulin G (IgG) in both phosphate buffered saline (PBS) and spiked in E. coli cell lysate. A superior target specificity for the sandwich assay compared to the corresponding direct assay is demonstrated along with a limit of detection of 90 pM in PBS. The prospect of improving the detection sensitivity was theoretically analysed, which indicated that the charge contrast between the target and the detection probe plays a crucial role in determining the signal. This aspect was then experimentally validated by modulating the zeta potential of the detection probe by conjugating negatively charged DNA oligonucleotides. The length of the conjugated DNA was varied from 5 to 30 nucleotides, altering the zeta potential of the detection probe from -9.3 ± 0.8 mV to -20.1 ± 0.9 mV. The measurements showed a clear and consistent enhancement of detection signal as a function of DNA lengths. The results presented here conclusively demonstrate the role of electric charge in detection sensitivity as well as the prospect for further improvement. The study therefore is a step forward in developing highly selective and sensitive electrokinetic assays for possible application in clinical investigations.
Topics: Biosensing Techniques; DNA; DNA Probes; Escherichia coli; Sensitivity and Specificity
PubMed: 33421763
DOI: 10.1016/j.bios.2020.112917 -
Talanta Jul 2022Analyte-sensitive DNA-based hydrogels find multiple applications in the field of biosensors due to their adaptable nature. Here, the design of DNA-based hydrogel and its...
Analyte-sensitive DNA-based hydrogels find multiple applications in the field of biosensors due to their adaptable nature. Here, the design of DNA-based hydrogel and its application as sensing platform for the detection of a specific target sequence are presented. DNA-functionalized hydrogel structures were formed via a free radical co-polymerization process. A simple one-step probe immobilization procedure is reported: DNA probe molecules are added to the photoactive polymer mixture, dispensed onto a solid support, or a mold, and covalently attached while the hydrogel is formed through UV light exposure. Such hydrogels can be synthesized with desired recognition ability through the selection of a certain nucleotide sequence. Here we show the application of DNA-based hydrogel to detect the target with high performance in fluorescence microarray format and, additionally, to fabricate holographic surface relief gratings for label-free sensing assays.
Topics: Biosensing Techniques; DNA; DNA Probes; Hydrogels; Microarray Analysis
PubMed: 35390683
DOI: 10.1016/j.talanta.2022.123427 -
Biosensors Jun 2023DNA-mediated nanotechnology has become a research hot spot in recent decades and is widely used in the field of biosensing analysis due to its distinctive properties of... (Review)
Review
DNA-mediated nanotechnology has become a research hot spot in recent decades and is widely used in the field of biosensing analysis due to its distinctive properties of precise programmability, easy synthesis and high stability. Multi-mode analytical methods can provide sensitive, accurate and complementary analytical information by merging two or more detection techniques with higher analytical throughput and efficiency. Currently, the development of DNA-mediated multi-mode analytical methods by integrating DNA-mediated nanotechnology with multi-mode analytical methods has been proved to be an effective assay for greatly enhancing the selectivity, sensitivity and accuracy, as well as detection throughput, for complex biological analysis. In this paper, the recent progress in the preparation of typical DNA-mediated multi-mode probes is reviewed from the aspect of deoxyribozyme, aptamer, templated-DNA and G-quadruplex-mediated strategies. Then, the advances in DNA-mediated multi-mode analytical methods for biological samples are summarized in detail. Moreover, the corresponding current applications for biomarker analysis, bioimaging analysis and biological monitoring are introduced. Finally, a proper summary is given and future prospective trends are discussed, hopefully providing useful information to the readers in this research field.
Topics: Biosensing Techniques; DNA; Nanotechnology; DNA Probes; Oligonucleotides
PubMed: 37504092
DOI: 10.3390/bios13070693 -
Bioconjugate Chemistry Sep 2022Fluorescent DNA probes were prepared in a modular approach using the "click" post-synthetic modification strategy. The new glycol-based module and DNA building block...
Fluorescent DNA probes were prepared in a modular approach using the "click" post-synthetic modification strategy. The new glycol-based module and DNA building block place just two carbons between the phosphodiester bridges and anchor the dye by an additional alkyne group. This creates a stereocenter in the middle of this artificial nucleoside substitute. Both enantiomers and a variety of photostable cyanine-styryl dyes as well as thiazole orange derivatives were screened as "clicked" conjugates in different surrounding DNA sequences. The combination of the ()-configured DNA anchor and the cyanylated cyanine-styryl dye shows the highest fluorescence light-up effect of 9.2 and a brightness of approximately 11,000 M cm. This hybridization sensitivity and fluorescence readout were further developed utilizing electron transfer and energy transfer processes. The combination of the hybridization-sensitive DNA building block with the nucleotide of 5-nitroindole as an electron acceptor and a quencher increases the light-up effect to 20 with the DNA target and to 15 with the RNA target. The fluorescence readout could significantly be enhanced to values between 50 and 360 by the use of energy transfer to a second DNA probe with commercially available dyes, like Cy3.5, Cy5, and Atto590, as energy acceptors at the 5'-end. The latter binary probes shift the fluorescent readout from the range of 500-550 nm to the range of 610-670 nm. The optical properties make these fluorescent DNA probes potentially useful for RNA imaging. Due to the strong light-up effect, they will not require washing procedures and will thus be suitable for live-cell imaging.
Topics: Alkynes; DNA; DNA Probes; Fluorescent Dyes; Glycols; Nucleosides; Nucleotides; RNA
PubMed: 35995426
DOI: 10.1021/acs.bioconjchem.2c00241 -
The Journal of Histochemistry and... Aug 2018Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal...
Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphases simultaneously, especially in single cells. Here, we present the selection of human probe DNA and a technique using multistep fluorescence in situ hybridization (FISH) employing four sets of six labeled FISH probes able to delineate all 24 human chromosomes in interphase cells. This full karyotype analysis approach will provide additional diagnostic potential for single cell analysis. The use of spectral imaging (SIm) has enabled the use of up to eight different fluorochrome labels simultaneously. Thus, scoring can be easily assessed by visual inspection, because SIm permits computer-assigned and distinguishable pseudo-colors to each probe during image processing. This enables full karyotype analysis by FISH of single-cell interphase nuclei.
Topics: Aneuploidy; Chromosomes, Artificial, Bacterial; DNA Probes; Humans; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Interphase; Karyotype; Karyotyping; Male; Plasmids; Single-Cell Analysis
PubMed: 29672206
DOI: 10.1369/0022155418771613 -
Scientific Reports Sep 2019Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of...
Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.
Topics: Costs and Cost Analysis; DNA Probes; DNA, Single-Stranded; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Thrombin
PubMed: 31558745
DOI: 10.1038/s41598-019-50371-1 -
Molecules (Basel, Switzerland) Dec 2022The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an...
The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g., homopurine or short mixed-sequence targets, low ionic strength, fully modified probe strands). Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides, are predisposed to recognize DNA targets due to their labile nature and high affinity towards complementary DNA. Here, we set out to gain further insight into the design parameters that impact the thermal denaturation properties and binding affinities of Invader probes. Towards this end, ten Invader probes were designed, and their biophysical properties and binding to model DNA hairpins and chromosomal DNA targets were studied. A Spearman's rank-order correlation analysis of various parameters was then performed. Densely modified Invader probes were found to result in efficient recognition of chromosomal DNA targets with excellent binding specificity in the context of denaturing or non-denaturing fluorescence in situ hybridization (FISH) experiments. The insight gained from the initial phase of this study informed subsequent probe optimization, which yielded constructs displaying improved recognition of chromosomal DNA targets. The findings from this study will facilitate the design of efficient Invader probes for applications in the life sciences.
Topics: In Situ Hybridization, Fluorescence; DNA; Oligonucleotides; Nucleotides; DNA, Complementary; DNA Probes
PubMed: 36615321
DOI: 10.3390/molecules28010127 -
Cytometry. Part a : the Journal of the... Dec 2019Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable...
Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a ~2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20°C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Topics: Animals; Biological Specimen Banks; Chromosomes; DNA Probes; Humans; In Situ Hybridization, Fluorescence; Microdissection
PubMed: 31532073
DOI: 10.1002/cyto.a.23896 -
Biophysical Journal Sep 2020Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology,...
Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology, and nanotechnology applications. A common approach relies on the immobilization on a surface of single-stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modeling of the same interactions on a surface are still challenging and perceived as strongly system dependent. Here, we show that a two-dimensional analysis of the kinetics of hybridization, performed at different target concentrations and probe surface densities by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behavior. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables quantifying the repulsive potential at the surface, as well as retrieving the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a three-dimensional immobilization strategy of probe strands with a double-stranded linker.
Topics: DNA; DNA Probes; DNA, Single-Stranded; Kinetics; Nucleic Acid Hybridization
PubMed: 32738217
DOI: 10.1016/j.bpj.2020.07.016 -
Viruses Jan 2021High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV...
High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 10 copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 10 plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.
Topics: Alphapapillomavirus; Cell Line, Tumor; DNA Probes, HPV; DNA, Viral; Genotype; Human Papillomavirus DNA Tests; Humans; Nucleic Acid Hybridization; Polymerase Chain Reaction; Software
PubMed: 33513748
DOI: 10.3390/v13020188