A nucleic acid dye-enhanced electrochemical biosensor for the label-free detection of Hg2+ based on a gold nanoparticle-modified disposable screen-printed electrode.

In this paper, a nucleic acid dye-enhanced electrochemical biosensor based on a screen-printed carbon electrode (SPCE) modified with Au nanoparticles (AuNPs) was designed for the detection of Hg2+ in water. AuNPs were modified on the surface of the disposable SPCE through the electrodeposition of HAuCl4. Subsequently, thiolated DNA probes were immobilized on the AuNP-modified electrode surface by Au-S reaction. After Hg2+ was bound with a DNA probe by thymine (T)-Hg2+-thymine (T) mismatch, the DNA probe was folded into a hairpin structure where positively charged GelRed molecules were embedded into the double-stranded part of the hairpin. Thus, the current of [Fe(CN)6]3-/4- increased significantly on account of the decreased electrostatic repulsion at the electrode surface. Under the optimized experimental conditions, the peak current of [Fe(CN)6]3-/4- exhibited a good linear relationship with lgCHg2+ in the concentration of Hg2+ linear range of 0.1 nM to 500 nM, and the limit of detection (S/N = 3) was calculated as 0.04 nM. The electrochemical sensor also exhibited excellent selectivity for Hg2+ in the presence of nine interfering ions, including Na+, Fe3+, Ni2+, Mg2+, Co2+, Pb2+, K+, Al3+ and Cu2+. Meanwhile, the developed electrochemical sensor was tested in the analysis of Hg2+ in tap water and river water samples, and the recoveries ranged from 81.0 to 114%. Therefore, this nucleic acid dye-enhanced electrochemical biosensor provided the advantages of simplicity, sensitivity, and specificity and is expected to be an alternative for Hg2+ detection in actual environmental samples.

One-Step Immobilization of Antibodies and DNA on Gold Sensor Surfaces via a Poly-Adenine Oligonucleotide Approach.

Label-free plasmonic biosensors have demonstrated promising capabilities as analytical tools for the detection of virtually any type of biomarker. They are presented as good candidates for precision diagnostics since they offer highly sensitive, cost-effective solutions that can be used in any clinical or laboratory setting without the need for specialized trainees. However, different surface functionalization protocols are required, depending on the nature of the biorecognition element, limiting their capabilities for integrated multi-biomarker detection. Here, we present a simple, yet efficient, one-step immobilization approach that is common for both DNA probes and antibodies. Our immobilization approach relies on the incorporation of poly-adenine (polyA) blocks in both nucleic acid probes and antibodies. PolyA sequences have a remarkable affinity for gold surfaces and can specifically interact with sufficient strength to generate stable, dense, and highly ordered monolayers. We have demonstrated excellent performance of our universal functionalization method, showing limits of detection and quantification in the pM-nM range. Moreover, it was able to reduce up to 50% of the background signal from undiluted serum samples compared to conventional methods, demonstrating the immense potential of this strategy for the direct analysis of human biofluids, essential for rapid point-of-care diagnostics. The polyA-based immobilization approach is a promising alternative for the generation of multiplexed biosensors that can detect both protein and nucleic acid biomarkers for multiparametric diagnostic assays.

Delayed Laboratory Response to COVID-19 Caused by Molecular Diagnostic Contamination.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries.

Ultrasensitive detection of nucleic acids using deformed graphene channel field effect biosensors.

Field-effect transistor (FET)-based biosensors allow label-free detection of biomolecules by measuring their intrinsic charges. The detection limit of these sensors is determined by the Debye screening of the charges from counter ions in solutions. Here, we use FETs with a deformed monolayer graphene channel for the detection of nucleic acids. These devices with even millimeter scale channels show an ultra-high sensitivity detection in buffer and human serum sample down to 600 zM and 20 aM, respectively, which are ∼18 and ∼600 nucleic acid molecules. Computational simulations reveal that the nanoscale deformations can form 'electrical hot spots' in the sensing channel which reduce the charge screening at the concave regions. Moreover, the deformed graphene could exhibit a band-gap, allowing an exponential change in the source-drain current from small numbers of charges. Collectively, these phenomena allow for ultrasensitive electronic biomolecular detection in millimeter scale structures.

Thermodynamics-Guided Strand-Displacement-Based DNA Probe for Determination of the Average Methylation Levels of Multiple CpG Sites.

Determination of the methylation levels of genes of interest is fundamental for biological and medical research that involves DNA methylation. Using the average methylation levels of multiple CpG sites to represent the methylation levels of the whole gene is much more accurate than using that of only one CpG site. However, current methods that can provide the average methylation levels of several CpG sites are all expensive, time-consuming (several weeks), and labor-intensive. Herein, guided by the unique thermodynamics of the DNA strand-displacement process, we constructed a DNA fluorescent probe for determination of the average methylation levels of multiple CpG sites. Theoretical calculations of the reaction process and proof-of-concepts experiments on two to three CpG sites of synthesized DNA validated the basic principles of our probe. Taking two CpG sites in the promotor regions of SF-1 (steroidogenic factor 1) gene and VDR (vitamin D receptor) gene as the targets, we successfully measured their average methylation levels in nine endometrial cancer patients and two healthy persons. We believe our probe will be a very useful tool in the field, and we anticipate it being widely adopted by biological and medical investigators.

A distinguished cancer-screening package containing a DNA sensor and an aptasensor for early and certain detection of acute lymphoblastic leukemia.

A disposable package of biosensors was developed along with the corresponding guidelines for early detection of the acute lymphoblastic leukemia cancer. This proposed cancer-screening package included a DNA sensor and an aptasensor as two main types of biosensors. The biosensors were used simultaneously. This combination of sensors can detect not only the presence of mutant genes but also the biomarkers of cancer. At current work, the combination of sensors were used to detect the presence of BCR-ABL1 as a mutant gene and CEA as a biomarkers of cancer, such a capability makes the package liable for early and certain detection of acute lymphoblastic leukemia. To construct both the DNA sensor and the aptasensor, a nanocomposite consisting of electrosynthesis carbon quantum dots and biosynthesized gold nanoparticles was applied. The construction of these biosensors was characterized using four different electrochemical methods including DPV (Differential Pulse Voltammetry), EIS (Electrochemical Impedance Spectroscopy), CV (Cyclic Voltammetry) and chronoamperometry. The peak current of a catechol solution that was used as an electroactive probe on the biosensor was linearly related to the logarithm of the concentrations of the target DNA and the target antigen in the range of 10 pM to 100 µM and 1 pg mL-1 to 0.001 g mL-1 with the detection limits of 1.5 pM and 0.26 pg mL-1 respectively, which are quite good results.

TiO2 nanowires as an effective sensing platform for rapid fluorescence detection of single-stranded DNA and double-stranded DNA.

Numerous nanomaterials have been utilized for novel biosensors with sensitivity and selectivity in the last decades due to their intrinsic unique properties. Herein, a facile fluorescence method for nucleic acid detection was developed by employing TiO2 nanowires (NWs) as the sensing platform. The quenching effect of TiO2 NWs to fluorophore-labelled single-stranded DNA (ssDNA) was found to be more significant than that to fluorophore-labelled double-stranded DNA (dsDNA) or triplex DNA probes. More importantly, the whole quenching process was also fast since it just took about ten minutes to reach the equilibrium. Based on the different affinities of TiO2 NWs to ssDNA, dsDNA and triplex DNA probes, the sequence-specific nucleic acids were detected with sensitivity and specificity. Further investigation has demonstrated that the quenching efficiency of TiO2 NWs to long ssDNA was apparently superior than that to short ssDNA. Moreover, the fluorescence from various ssDNA probes labelled with a wide spectrum of fluorescent dyes could also be quenched by TiO2 NWs. These inspiring results reveal that TiO2 NWs could be an excellent universal nanoquencher used in the next-generation biosensors.

Enhanced Sensitivity in Nanopore Sensing of Cancer Biomarkers in Human Blood via Click Chemistry.

Cancer biomarkers are expected to be indicative of the occurrence of certain cancer diseases before the tumors form and metastasize. However, many biomarkers can only be acquired in extremely low concentrations, which are often beyond the limit of detection (LOD) of current instruments and technologies. A practical strategy for nanopore sensing of cancer biomarkers in raw human blood down to the femtomolar level is developed here. This strategy first converts the detection of cancer biomarkers to the quantification of copper ions by conducting a sandwich assay involving copper oxide nanoparticles. The released Cu2+ is then taken to catalyze the "click" reaction which ligates a host-guest modified DNA probe. Finally, this DNA probe is subjected to single-channel recordings to afford the translocation events that can be used to derive the concentrations of the original biomarkers. Due to the amplification effects of nanoparticle loadings and the "click" reaction, the LOD of this strategy can be as low as the subfemtomolar level. Further, the acid treatment step could effectively eliminate the interferences from plasma proteins in raw human blood and make the strategy highly suitable for the detection of cancer biomarkers in clinical samples.

A Non-Label and Enzyme-Free Sensitive Detection Method for Thrombin Based on Simulation-Assisted DNA Assembly.

Taking advantage of the high selectivity of aptamers and enzyme-free catalyzed hairpin assembly (CHA) amplification strategy, we herein describe a label-free and enzyme-free sensitive fluorescent and colorimetric strategy for thrombin detection in this paper. In the presence of target, the corresponding aptamer of the partial dsDNA probes will bind to the target and liberate the initiation strand, which is artfully designed as the “on” switch for hairpin assembly. Moreover, the displaced initiation strand partakes in a multi-cycle process and produces numerous G-quadruplexes, which have a remarkable enhancement in fluorescent/colorimetric signal from NMM (N-methyl-mesoporphyrin IX) and TMB (3,3′,5,5′-tetramethylbenzidine), respectively. The proposed amplification strategy for thrombin detection is of high sensitivity, down to 2.4 pM, and also achieves colorimetric signals that are able to be distinguished by naked eye. More importantly, the thermodynamics of interacting DNA strands used in our work, and the process of toehold strand displacement-driven assembly are simulated before biological testing, verifying the feasibility theoretically, and simplifying the subsequent actual experiments. Therefore, our approach and simulation have a certain potential application in biomarker detection and quantitatively monitor for disease diagnosis.

Gold nanoparticle-based 2'-O-methyl modified DNA probes for breast cancerous theranostics.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulated diverse cellular processes including differentiation, proliferation, apoptosis, metabolism and signal transduction pathways. An increasing number of data suggested that miRNA-21 could be identified as diagnostic and therapeutic biomarker for breast cancer. Meanwhile, inhibiting the function of miRNA-21, resulting in cells growth inhibition and apoptotic cells death. To realize miRNA-21detection and inhibition to diagnostic and therapeutic breast cancer cells, we developed gold nanoparticle-based 2'-O-methyl modified DNA probes (AuNP-2'-OMe-DNA probes) for diagnostic and therapeutic breast cancer. Gold nanoparticles were functionalized with chemically modified miRNA-21 inhibitor to suppress the function of miRNA-21 for the therapeutic breast cancer, at the same time, fluorophore-labeled DNA molecules were hybridized with antimiRNA-21 for diagnostic breast cancer. The results showed that the 2'-O-methyl modified DNA can improve stability, increase binding affinity to target strands and enhance the therapeutic effects. The experimental results also demonstrated that antimiR-21 were efficiently introduced into the cells and knocked down miRNA-21 to inhibit its function, leading to growth inhibition and apoptotic cells death. We prospected that chemically modified miRNA-21 inhibitor based on gold nanoparticles would be as a promising diagnostic and therapeutic platform for breast cancer clinically.

Microfluidic System for Detection of Viral RNA in Blood Using a Barcode Fluorescence Reporter and a Photocleavable Capture Probe.

A microfluidic sample preparation multiplexer (SPM) and assay procedure is developed to improve amplification-free detection of Ebola virus RNA from blood. While a previous prototype successfully detected viral RNA following off-chip RNA extraction from infected cells, the new device and protocol can detect Ebola virus in raw blood with clinically relevant sensitivity. The Ebola RNA is hybridized with sequence specific capture and labeling DNA probes in solution and then the complex is pulled down onto capture beads for purification and concentration. After washing, the captured RNA target is released by irradiating the photocleavable DNA capture probe with ultraviolet (UV) light. The released, labeled, and purified RNA is detected by a sensitive and compact fluorometer. Exploiting these capabilities, a detection limit of 800 attomolar (aM) is achieved without target amplification. The new SPM can run up to 80 assays in parallel using a pneumatic multiplexing architecture. Importantly, our new protocol does not require time-consuming and problematic off-chip probe conjugation and washing. This improved SPM and labeling protocol is an important step toward a useful POC device and assay.

Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay.

BACKGROUND: Mutation of epidermal growth factor receptor (EGFR) is a prediction marker of the response to tyrosine kinase inhibitor (TKI) drugs in non-small cell lung cancer (NSCLC) patients. As late stage lung cancer patients rarely undergo surgery, samples for EGFR mutation identification usually come from computed tomography (CT)-guided or endoscopic biopsies, which is invasive and costly. Pleural effusion may serve as a less invasive sample for EGFR mutation detection. METHODS: We designed a fluorophore-labeled peptide nucleic acid (PNA) probe assay for three types of EGFR mutations, including exon 19 deletions, L858R point mutations and T790M point mutations. The assay was applied in 39 pleural effusion samples from NSCLC patients. The correlation between detected EGFR status and clinical outcome were analyzed. RESULTS: In 15 paired samples, PNA probe assay in pleural effusion samples could detect all the mutations that were identified by conventional PCR plus Sanger sequencing in tissue biopsies. In addition, PNA probe assay detected three more T790M mutations. In all 39 pleural effusions, the PNA probe assay detected 27 having at least one of the three EGFR mutations. Among the patients before TKI treatment, those with a sensitizing mutation (either exon 19 deletion or L858R) but without T790M, had 94.1% response rate and longer progression-free survival (mean 10.8 months) than patients without detected mutation (mean 4.2 months) and patients with T790M (mean 1.7 months). CONCLUSIONS: Mutations detected in pleural effusions using PNA probe assay are highly associated with clinical outcome. This method appears to be a reliable way for the prediction of the efficacy of EGFR-targeted therapy.

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10-15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles.

DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.

Simultaneous Identification of 13 Foodborne Pathogens by Using Capillary Electrophoresis-Single Strand Conformation Polymorphism Coupled with Multiplex Ligation-Dependent Probe Amplification and Its Application in Foods.

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) coupled with stuffer-free multiplex ligation-dependent probe amplification (MLPA) was developed to identify 13 species of foodborne pathogens simultaneously. Species-specific MLPA probes were designed for nine of these species. These probes were targeted to the groEL, glyA, MMS, tuf, inv, ipaH, nuc, vvh, and 16S rRNA genes, which corresponded to Bacillus cereus, Campylobacter coli, Cronobacter sakazakii, Enterococcus spp., Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio vulnificus, and Yersinia enterocolitica, respectively. MLPA probes that had been previously developed by our laboratory were used for the other four species (Campylobacter jejuni, Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes). The CE-SSCP method was optimized to identify all 13 foodborne microbes simultaneously in a single electrogram, in which 50-500 pg genomic DNA was detected per microbe. Twelve species were detected from animal-derived food samples (specifically, milk and sliced ham) that had been artificially inoculated with 12 of the foodborne pathogens, excluding V. vulnificus, which is not usually associated with animal foods. The method developed here could be used as an early warning system for outbreaks of foodborne diseases associated with animal-derived foods in the food industry.

Sensitive detection of multiple pathogens using a single DNA probe.

A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP.

Colorimetric Detection by Gold Nanoparticle DNA Probes for Miltenberger Series (GP.Mur, GP.Hop, and GP.Bun) Identification.

BACKGROUND: Miltenberger (Mi) series are the collective glycophorin hybrids in the MNS blood group system. Mi series are composed of several subtypes, for examples, GP.Mur, GP.Hop, and GP.Bun. The incompatibility of Mi series blood transfusion poses the risk of hemolysis. Due to the lack of standard antibodies for Mi series blood typing, colorimetric gold nanoparticle (AuNP) DNA probes were therefore explored for Mi series identification. METHODS: AuNPs were synthesized and conjugated to an RvB (test) probe and an RvA2 (control) probe. Each of the AuNP DNA probes was tested against the amplified products of Mi(+) (GP.Mur/Hop/Bun), Mi(-), and the blank (no amplified product). The change in color was observed by visual inspection and UV-Vis spectroscopy. RESULTS: The amplified product of the Mi(+) sample retained the color on both probes (test+/control+). The amplified product of the Mi(-) sample retained the color only on the control probe (test-/control+) and the amplified product of the blank turned clear on both probes (test-/control-). The results by optical density absorbance measurement were concordant with the results by visual inspection. Both probes were validated with the amplified products of the ten Mi(+) and ten Mi(-) samples. All of the samples were correctly identified. CONCLUSION: AuNP DNA probes (RvB and RvA2) could be applied to distinguish the amplified products of Mi(+), Mi(-), and the blank by visual inspection and/or OD absorbance measurement.

Deoxyribonucleic Acid Probes Analyses for the Detection of Periodontal Pathogens.

BACKGROUND: In clinical microbiology several techniques have been used to identify bacteria. Recently, Deoxyribonucleic acid (DNA)-based techniques have been introduced to detect human microbial pathogens in periodontal diseases. Deoxyribonucleic acid probes can detect bacteria at a very low level if we compared with the culture methods. These probes have shown rapid and cost-effective microbial diagnosis, good sensitivity and specificity for some periodontal pathogens in cases of severe periodontitis. MATERIALS AND METHODS: Eighty-five patients were recruited for the study. Twenty-one subjects ranging between 22 and 48 years of age fulfilled the inclusion and exclusion criteria. Seventy-eight samples became available for DNA probe analysis from the deepest pockets in each quadrant. RESULTS: All 21 patients showed positive results for Prevotella intermedia; also, Prevotella gingivalis was identified in 19 subjects, Aggregatibacter actinomycetemcomitans in 6 subjects. P. intermedia was diagnosed positive in 82% of the subgingival samples taken, 79% for P. gingivalis, and 23% for A. actinomycetemcomitans. CONCLUSION: This study shows a high frequency of putative periodontal pathogens by using DNA probe technology, which is semi-quantitative in this study. Deoxyribonucleic acid probes can detect bacteria at very low level about 10(3) which is below the detection level of culture methods. The detection threshold of cultural methods. CLINICAL SIGNIFICANCE: The three types of bacteria can be detected rapidly with high sensitivity by using the DNA probe by general practitioners, and thus can help in the diagnosis process and the treatment.

Real-time reverse transcriptase polymerase chain reaction assays for Middle East Respiratory Syndrome.

Here we designed and tested two highly specific quantitative TaqMan(®)-MGB-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays for Middle East Respiratory Syndrome (MERS). The primers and probes for these assays were evaluated and found to have a limit of detection (LOD) of 0.005 plaque-forming units/PCR (pfu/PCR).