DNA Probes Research Articles

Dual-Stimuli Regulation of DNAzyme Cleavage Reaction by Coordination-Driven Nanoprobes for Cancer Cell Imaging.

Endowing current artificial chemical reactions (ACRs) with high specificity and intricate activation capabilities is crucial for expanding their applications in accurate bioimaging within living cells. However, most of the reported ACR-based evaluations relied on either single biomarker stimuli or dual activators without obvious biological relevance, still limiting their accuracy and fidelity. Herein, taking the metal-ion-dependent DNAzyme cleavage reaction as a model ACR, two regulators, glutathione (GSH) and telomerase (TE) activated DNAzyme cleavage reactions, were exploited for precise discrimination of cancerous cells from normal cells. DNA probe was self-assembled into the ZIF-90 nanoparticle framework to construct coordination-driven nanoprobes. This approach enhances the stability and specificity of tumor imaging by utilizing biomarkers associated with rapid tumor proliferation and those commonly overexpressed in tumors. In conclusion, the research not only paves the way for new perspectives in cell biology and pathology studies but also lays a solid foundation for the advancement of biomedical imaging and disease diagnostic technologies.

A New SPQC Biosensor for the Detection of a New Target of Escherichia/Shigella Genera Based on a Novel Method of Synthesizing Long-Range DNA.

The rapid and sensitive detection of Escherichia/Shigella genera is crucial for human disease and health. This study introduces a novel series of piezoelectric quartz crystal (SPQC) sensors for detecting Escherichia/Shigella genera. In this innovative biosensor, we propose a new target and novel method for synthesizing long-range DNA. The method relies on the amplification of two DNA probes, referred to as H and P amplification (HPA), resulting in the products of long-range DNA named Sn. The new target was screened from the 16S rRNA gene and utilized as a biomarker. The SPQC sensor operates as follows: the Capture probe is modified on the electrodes. In the presence of a Displace probe and target, the Capture can form a complex with the Displace probe. The resulting complex hybridizes with Sn, bridging the gap between the electrodes. Finally, silver wires are deposited between the electrodes using Sn as a template. This process results in a sensitive response from the SPQC. The detection limit of the SPQC sensor is 1 CFU/mL, and the detection time is within 2 h. This sensor would be of great benefit for food safety monitoring and clinical diagnosis.

Label-free detection of platelet-derived growth factor-BB via liquid crystal/aqueous interface functionalized using a double-stranded DNA probe

A liquid crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with cationic surfactant octyltrimethylammonium bromide (OTAB) and adsorbed with a double-stranded deoxyribonucleic acid (dsDNA) probe at the LC/aqueous interface (TEMOTAB/DNA) was utilized for the highly specific detection of platelet-derived growth factor (PDGF-BB). Upon adsorption of the OTAB-coated 4-cyano-4′-pentylbiphenyl (5CB) on the TEM grid (TEMOTAB) and subsequent adsorption of the dsDNA probe, a homeotropic orientation was observed. Subsequently, the TEMOTAB/DNA biosensor was exposed to PDGF-BB, which induced a homeotropic-to-planar configurational change in 5CB, as observed using polarized optical microscopy with crossed polarizers. The optimized adsorption density of the dsDNA probe facilitated the detection of 2.63 pg mL−1 PDGF-BB in the detection range of 5–200 pg mL−1. Remarkably, this LC-based biosensor discriminated PDGF-BB from bovine serum albumin, immunoglobulin G, hemoglobin, carcinoembryonic antigen, epidermal growth factor, PDGF-AA, and PDGF-AB. The application potential of this sensor was further demonstrated by its effective performance in detecting PDGF-BB in both human serum and synthetic urine samples. Because of its high specificity, sensitivity, and label-free detection capabilities, this biosensor has potential applications in cancer detection.

Re-Examination of PGT-A Detected Genetic Pathology in Compartments of Human Blastocysts: A Series of 23 Cases.

Background: In recent years, preimplantation genetic testing for aneuploidies (PGT-A) has become widespread in assisted reproduction. However, contrary to expectations, PGT-A does not significantly improve the clinical outcomes of assisted reproductive technologies. One of the underlying reasons is the discordance between the PGT-A results and the true chromosomal constitution of the blastocyst. In this case series, we re-examined the PGT-A results in trophectoderm (TE) re-biopsies and in the two isolated blastocyst compartments-the TE and the inner cell mass (ICM). Methods: This study enrolled 23 human blastocysts from 17 couples who were referred for assisted reproduction. The blastocysts were unsuitable for uterine transfer due to the chromosomal imbalance revealed by PGT-A using array comparative genomic hybridization (aCGH) (n = 11) or next-generation sequencing (NGS) (n = 12). The re-examination of the PGT results involved two steps: (1) a TE re-biopsy with subsequent aCGH and (2) blastocyst separation into the TE and the ICM with a subsequent cell-by-cell analysis of each isolated compartment by fluorescence in situ hybridization (FISH) with the DNA probes to chromosomes 13, 16, 18, 21, and 22 as well as to the PGT-A detected imbalanced chromosomes. Results: In 8 out of 23 cases, the PGT-A results were concordant with both the re-biopsy and the isolated TE and ICM analyses. The latter included the diagnoses of full non-mosaic aneuploidies (five cases of trisomies and two cases of monosomies). In one case, the results of PGT-A, aCGH on the TE re-biopsy, and FISH on the isolated TE showed Xp tetrasomy, which contrasted with the FISH results on the isolated ICM, where this chromosomal pathology was not detected. This case was classified as a confined mosaicism. In 4 out of 23 cases, the results were partially discordant. The latter included one case of trisomy 12, which was detected as non-mosaic by PGT-A and the re-biopsy and as mosaic by FISH on the isolated TE and ICM. This case was classified as a true mosaicism with a false negative PGT-A result. In 11 out of 23 cases, the re-examination results were not concordant with the PGT-A results. In one of these discordant cases, non-mosaic tetraploidy was detected by FISH in the isolated TE and ICM, whereas the PGT-A and the TE re-biopsy failed to detect any abnormality, which advocated for their false negative result. In two cases, the re-examination did not confirm full aneuploidies. In eight cases, full or partial mosaic aneuploidies as well as chaotic mosacism were not confirmed in the isolated TE nor the isolated ICM. Thus, in 47.8% of cases, the PGT-A results did not reflect the true chromosomal constitution of a blastocyst. Conclusions: The PGT results may have different prognostic value in the characterization of the chromosomal constitution of a blastocyst. The detected non-mosaic aneuploidies have the highest prognostic value. In stark contrast, most PGT-identified mosaic aneuploidies fail to characterize the true chromosomal constitution of a blastocyst. Once detected, a differential diagnosis is needed.

Rapid and Label-Free Detection of Influenza A Virus With a SAW Biosensor Based on DNA Probes

Rapid and Label-Free Detection of Influenza A Virus With a SAW Biosensor Based on DNA Probes

Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing

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.

Construction of Multiply Guaranteed DNA Sensors for Biological Sensing and Bioimaging Applications.

Nucleic acids exhibit exceptional functionalities for both molecular recognition and catalysis, along with the capability of predictable assembly through strand displacement reactions. The inherent programmability and addressability of DNA probes enable their precise, on-demand assembly and accurate execution of hybridization, significantly enhancing target detection capabilities. Decades of research in DNA nanotechnology have led to advances in the structural design of functional DNA probes, resulting in increasingly sensitive and robust DNA sensors. Moreover, increasing attention has been devoted to enhancing the accuracy and sensitivity of DNA-based biosensors by integrating multiple sensing procedures. In this review, we summarize various strategies aimed at enhancing the accuracy of DNA sensors. These strategies involve multiple guarantee procedures, utilizing dual signal output mechanisms, and implementing sequential regulation methods. Our goal is to provide new insights into the development of more accurate DNA sensors, ultimately facilitating their widespread application in clinical diagnostics and assessment.

Zeolite-iron oxide integrated interdigitated electrode sensor for diagnosing cervical cancer

Cervical cancer is caused by changes in the cervix that lead to precancerous cells and eventually progress to cancer. Human papillomavirus (HPV) infections are the primary cause of cervical cancer. Early detection of HPV is crucial in preventing cervical cancer, and regular screening for HPV infection can identify cell changes before they develop into cancer. While Pap smear tests are reliable for cervical cancer screening, they are critical, expensive, and labor-intensive. Therefore, researchers are focusing on identifying blood-based biomarkers using biosensors for cervical cancer screening. HPV strains 16, 45, and 18 are common culprits in cervical cancer. This study aimed to develop an HPV-16 DNA biosensor on a zeolite-iron oxide (zeolite-IO) modified interdigitated electrode (IDE) sensor. The DNA probe was immobilized on the IDE through amine-modified zeolite-IO, enhancing the hybridization of the target and DNA probe. The detection limit of the DNA-DNA duplex was found to be 7.5 pM with an R2 value of 0.9868. Additionally, control experiments with single and triple mismatched sequences showed no increase in current responses, and the identification of target DNA in a serum-spiked sample indicated specific and selective target identification.

Identifying SARS-CoV-2 Variants Using Single-Molecule Conductance Measurements.

The global COVID-19 pandemic has highlighted the need for rapid, reliable, and efficient detection of biological agents and the necessity of tracking changes in genetic material as new SARS-CoV-2 variants emerge. Here, we demonstrate that RNA-based, single-molecule conductance experiments can be used to identify specific variants of SARS-CoV-2. To this end, we (i) select target sequences of interest for specific variants, (ii) utilize single-molecule break junction measurements to obtain conductance histograms for each sequence and its potential mutations, and (iii) employ the XGBoost machine learning classifier to rapidly identify the presence of target molecules in solution with a limited number of conductance traces. This approach allows high-specificity and high-sensitivity detection of RNA target sequences less than 20 base pairs in length by utilizing a complementary DNA probe capable of binding to the specific target. We use this approach to directly detect SARS-CoV-2 variants of concerns B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron) and further demonstrate that the specific sequence conductance is sensitive to nucleotide mismatches, thus broadening the identification capabilities of the system. Thus, our experimental methodology detects specific SARS-CoV-2 variants, as well as recognizes the emergence of new variants as they arise.

Label-Free Ratiometric Homogeneous Electrochemical Strategy Based on Exonuclease III-Aided Signal Amplification for Facile and Rapid Detection of miR-378.

MiR-378 is abnormally expressed in various cancers, such as hepatocellular carcinoma, renal cell carcinoma, and nonsmall cell lung cancer. Here, we developed a label- and immobilization-free ratiometric homogeneous electrochemical strategy based on exonuclease III (Exo III) for the facile and rapid determination of miR-378. Two 3'-protruding hairpin DNA probes (HPs) are designed in this strategy. Doxorubicin (DOX) and potassium ferrocyanide (Fe2+) were used as label-free probes to produce a response signal (IDOX) and a reference signal (IFe2+) in the solution phase. When no target was present in the solution, the HP was stable, most of the DOX was intercalated in the stem of the HP, and the diffusion rate of DOX was significantly reduced, resulting in reduced electrochemical signal response. When miR-378 was present, double-cycle signal amplification triggered by Exo III cleavage was initiated, ultimately disrupting the hairpin structures of HP1 and HP2 and releasing a large amount of DOX into the solution, yielding a stronger electrochemical signal, which was low to 50 pM. This detection possesses excellent selectivity, demonstrating high application potential in biological systems, and offers simple and low-cost electrochemical detection for miR-378.

DNA tetrahedral molecular sieve for size-selective fluorescence sensing of miRNA 21 in living cells

In situ monitoring of intracellular microRNAs (miRNAs) often encounters the challenges of surrounding complexity, coexistence of precursor miRNAs (pre-miRNAs) and the degradation of biological enzyme in living cells. Here, we designed a novel probe encapsulated DNA tetrahedral molecular sieve (DTMS) to realize the size-selective detection of intracellular miRNA 21 that can avoid the interference of pre-miRNAs. In such strategy, quencher (BHQ-1) labeled probe DNA (S6-BHQ 1) was introduced into the inner cavity of fluorophore (FAM) labeled DNA tetrahedral scaffolds (DTS) to prepare DTMS, making the FAM and BHQ-1 closely proximate, and resulting the sensor in a "signal-off" state. In the presence of miRNA 21, strand displacement reaction happened to form more stable DNA double-stranded structure, accompanied by the release of S6-BHQ 1 from the inner cavity of DTMS, making the sensor in a "signal-on" state. The DTMS based sensing platform can then realized the size-selective detection of miRNA 21 with a detection limit of 3.6 pM. Relying on the mechanical rigidity of DTS and the encapsulation of DNA probe using DTMS, such proposed method achieved preferable reproducibility and storage stability. Moreover, this sensing system exhibited good performance for monitoring the change of intracellular miRNA 21 level during the treatment with miRNA-related drugs, demonstrating great potential for biological studies and accurate disease diagnosis.

Abstract A007: Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is encoded by the ERBB2 gene, and frequently mutated, amplified, and/or overexpressed in urothelial cancer (UC). Promising antibody-drug conjugates have led to new interest in HER2 as a UC clinical target. Patient selection for HER2-targeted therapy typically relies on immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of tumor tissue, however DNA sequencing can identify ERBB2 genomic alterations. Accurate biomarker-driven patient selection will be critical to optimize the clinical benefit of HER2-targeted therapy. Therefore, we evaluated plasma circulating tumor DNA (ctDNA) for ERBB2/HER2 status determination in UC, as compared to IHC and FISH of metachronous tissue. 411 plasma samples from 236 metastatic UC patients were profiled with targeted sequencing across >50 genes frequently altered in UC, including dense coverage flanking the ERBB2 locus. Using an established bioinformatics workflow, we determined the presence of ERBB2 alterations (mutations, amplifications, and/or structural variants) in the 181 patients with evidence of ctDNA in ≥1 sample. Clinical records were reviewed for the availability of archival tumor tissue; in total, 81 formalin-fixed paraffin-embedded tissue specimens were retrieved from 23 patients with ctDNA ERBB2 alterations and 20 ERBB2-wildtype controls. HER2 IHC was performed with a polyclonal rabbit anti-human Her2 antibody (Dako) and scored according to gastric cancer guidelines. HER2 FISH was performed with the PathVysion HER-2 DNA Probe Kit. Protein-altering ERBB2 mutations were identified in 14% of evaluable patients, with two-thirds at known oncogenic hotspots. ERBB2 copy gain was detected in 8% of patients overall, 9% when excluding low tumor fraction samples. IHC results were assessable for 82 tissue samples from 43 patients, and 33 patients had at least one sample with positive HER2 staining (2+/3+). IHC scores varied in 16/23 patients with ≥2 tissue samples, with the variation leading to a change in classification (HER2-negative [0/1+] versus positive [2+/3+]) in half (8/16). In two patients with mixed variant histology, the urothelial component showed IHC 3+ while the scores were 2+ (plasmacytoid) and 0 (squamous) in the variant regions. Frequent focal staining patterns were observed with both IHC and FISH. ERBB2 alterations in ctDNA were correlated with IHC positivity – when considering the most recent tissue sample per patient, 79% of ctDNA ERBB2-altered patient’s tumors were also positive by IHC. Conversely, 55% of ctDNA ERBB2-wildtype cases were positive by IHC. FISH results were available from 24 samples (20 positive, 4 negative), all from patients with ctDNA ERBB2 amplification. 18/20 samples with FISH positivity were HER2 IHC 2+/3+; the remaining two samples were IHC 1+. Our results demonstrate significant spatial and/or temporal heterogeneity in ERBB2/HER2 status, with implications for the rational implementation of biomarker-directed (HER2-targeted) therapy in UC. Citation Format: Gillian Vandekerkhove, Andrew J. Murtha, David C. Müller, Kimia Rostin, Carlos Vasquez-Rios, Jussi Nikkola, Maria Stephenson, Emily Fung, Jaskirat Atwal, Karan Parekh, Cecily Q. Bernales, Gráinne Donnellan, Gang Wang, Tilman Todenhöfer, Piet Ost, Peter C. Black, Kim N. Chi, Bernhard J. Eigl, Alexander W. Wyatt. Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A007.

Detection of fucosylated extracellular vesicles miR-4732-5p related to diagnosis of early lung adenocarcinoma by the electrochemical biosensor

Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg2+-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg2+-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π–π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10−17 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.

An electrophoretic mobility shift assay using the protein isolated from host plants

BackgroundThe electrophoretic mobility shift assay (EMSA) is a common technology to detect DNA-protein interactions. However, in most cases, the protein used in EMSA is obtained via prokaryotic expression, and rarely from plants. At the same time, the proteins expressed from prokaryotic systems usually cannot fold naturally and have no post translationally modification, which may affect the binding of proteins to DNA.ResultsHere, we develop a technique to quickly isolate proteins of interest from host plants and then analyze them using fluorescent EMSA. This technology system is called: protein from plants fluorescent EMSA method (PPF-EMSA). In PPF-EMSA, a special transient transformation method is employed to transiently deliver genes into the plant, enabling efficient synthesis the encoded proteins. Then, the target protein is isolated using immunoprecipitation, and the DNA probes were labeled with cyanine 3 (Cy3). Both fluorescent EMSA and super-shift fluorescent EMSA can be performed using the proteins from plants. Three kinds of plants, Betula platyphylla, Populus. davidiana×P. bolleana and Arabidopsis thaliana, are used in this study. The proteins isolated from plants are in a natural state, can fold naturally and are posttranslationally modified, enabling true binding to their cognate DNAs.ConclusionAs transient transformation can be performed quickly and not depended on whether stable transformation is available or not, we believe this method will have a wide application, enabling isolation of proteins from host plant conveniently.

The potential of tailed amplicons for SARS-CoV-2 detection in Nucleic Acid Lateral Flow Assays.

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/μL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.

Immobilization-free and label-free electrochemical DNA biosensing based on target-stimulated release of redox reporter and its catalytic redox recycling

Herein, we demonstrate a simple, homogenous and label-free electrochemical biosensing system for sensitive nucleic acid detection based on target-responsive porous materials and nuclease-triggered target recycling amplification. The Fe(CN)63− reporter was firstly sealed into the pores of Fe3O4 nanoparticles by probe DNA. Target DNA recognition triggered the controllable release of Fe(CN)63− for the redox reaction with the electron mediator of methylene blue enriched in the dodecanethiol assembled electrode and thereby generating electrochemical signal. The exonuclease III (Exo III)-assisted target recycling and the catalytic redox recycling between Fe(CN)63− and methylene blue contributed for the enhanced signal response toward target recognition. The low detection limit toward target was obtained as 478 fM and 1.6 pM, respectively, by square wave voltammetry and cyclic voltammetry methods. It also possessed a well-discrimination ability toward mismatched strands and high tolerance to complex sample matrix. The coupling of bio-gated porous nanoparticles, nuclease-assisted target amplification and catalytic redox recycling afforded the sensing system with well-controllable signal responses, sensitive and selective DNA detection, and good stability, reusability and reproducibility. It thus opens a new avenue toward the development of simple but sensitive electrochemical biosensing platform.

Development of a multiplex one-tube loop-mediated isothermal amplification method for rapidly simultaneous detection of the mecA and nuc genes in methicillin-resistant Staphylococcus aureus

This study developed a multiplex one-tube loop-mediated isothermal amplification method using fluorescent dye-labeled DNA probes (mfLAMP) for the rapid simultaneous detection of the nuc (S. aureus-specific marker) and mecA genes (PBP2a-specific marker) in methicillin-resistant Staphylococcus aureus (MRSA). In the mfLAMP method, a graphene oxide (GO) was used as an effective adsorbent to eliminate free fluorescent dye-labeled DNA probes (mecAf-probe: Cy5-ssDNA-Biotin and nucf-probe: FAM-ssDNA-Biotin) present in negative results. The samples were then measured using a developed program that enable simultaneous detection of fluorescein (FAM) and Cyanine5 (Cy5). Green fluorescence represents the detection of the nuc gene, red fluorescence indicates the detection of the mecA gene, and yellow fluorescence signifies the detection of MRSA. The mfLAMP was specific to MRSA and could detect 10°CFU/mL of MRSA in standard buffer and artificially inoculated irrigation water. The recovery rate from contaminated irrigation water ranged from 100.41 to 109.85 %. This study is the first to report the mfLAMP using GO, which could be point-of-care testing for the rapid and simultaneous detection of MRSA's nuc and mecA genes. The results demonstrate that the mfLAMP will be a novel tool for simultaneously detecting more than two target genes or bacteria within 1 h without an enrichment step.

Bimodal 3D DNA nanomachines coupling with bioconjugates powered electrochemical and visual-driven dual-enzyme cascade sensing of Let-7a

A dual-enzyme self-powered biosensor utilizing bimodal 3D DNA nanomachines and gold nanoparticle (AuNPs)/carbon/disulfide molybdenum hollow nanorods bioconjugates is developed for the electrochemical and visual detection of the tumor biomarker let-7a. DNA probes are immobilized on a flexible electrode substrate of AuNPs/graphdiyne/carbon cloth to improve probe loading capacity and electron transfer efficiency. The presence of let-7a initiates the work of dual-enzyme 3D DNA nanomachine, and the output DNA single strand cleaved by Nb.BbvCI is served as a bridge to connect glucose oxidase (GOD)-DNA conjugate. The GOD coordinates with the cathodic enzyme bilirubin oxidase and produces an increased open circuit voltage signal, which is attributed to the hexaammine ruthenium in the double-stranded adsorption system of the biocathode. By utilizing the H2O2 generated during the oxidation process of GOD, a linear relationship between the color of 3,3′,5,5′-tetramethylbenzidine and the concentration of let-7a is established, enhancing the reliability of the detection results through the independent signal conversion mechanisms of electrochemical/colorimetric modes. The results demonstrate a wide dynamic range of 0.0001–0.1 pM with detection limits of 31.04 aM (S/N=3) in electrochemical mode and 59.99 aM (S/N=3) in the colorimetric mode. This dual-mode biosensing strategy can be expanded to detect additional nucleic acid biomarkers, providing a promising support and reference for biological analysis and medical diagnosis.

Abstract PO4-26-05: Dual HER2 Testing With Both Immunohistochemistry (IHC) and ISH (In Situ Hybridization): Long Term Analysis of Dual Testing in a Single Academic Institution

Abstract Background: HER-2 critically guides therapies and is prognostic in breast cancer (BC). Current ASCO/CAP guidelines for immunohistochemistry (IHC) define 3+ as positive, 0 and 1+ as negative, and 2+ as equivocal, with additional testing recommended. Current guidelines for in situ hybridization (ISH) define a HER-2/CEP17 ratio of ≥2.0, with an average HER-2 copy number of ≥4.0 signals/cell as ISH positive. With < 4.0 copies HER-2/cell, and for various other ISH groups that are borderline, further specific additional testing is recommended. Data suggest that HER-2 copy number does have clinical relevance in HER-2+ BC, however most clinical trials evaluating HER-2 targeted therapeutics have defined HER-2+ as specifically a HER-2/CEP17 ratio ≥2.0. Limited studies have explored HER-2 copy number and its clinical implication in the HER-2 1+, and 2+ ISH negative population, recently described as HER-2 low. Further, the guidelines for HER-2 testing have changed twice over the past decade, and little is known about the impact of these changes. Design: From 2001- March 2023, all new invasive BC cases tested at University of Vermont (UVM) for HER-2 with both IHC and ISH were analyzed. All testing was performed on formalin-fixed paraffin-embedded tissue, using the HercepTest kit (Dako Corp, Carpinteria, California) (2001-2012) 4B5 Rabbit Monoclonal Antibody (Ventana) (2013-2023) for IHC and the INFORM dual ISH DNA probe (Ventana) for CISH. Interpretation was performed using published criteria at the time of original testing, and all cases with both IHC and CISH testing were independently analyzed by two experienced reviewers. Results: 2734 cases had dual testing with IHC and CISH. 2407 patients (88%) had a HER-2/CEP17 ratio < 2.0, of these, 6.4% had a HER-2 copy number ≥4 and < 6. Only 12% of cases overall had a ratio ≥ 2.0. In IHC 0 and 1+ cases with HER-2/CEP17 ratio of < 2, 0.4% and 4.3% have excess HER-2 copies/cell (defined as ≥ 4), respectively, compared to 13.8% for IHC 2+. For IHC 2+ cases, only 13.6% overall demonstrate amplification, with however, again, an additional 13.8% having excess copy number. Interestingly, 9.6% of the IHC 3+ cases had a ratio of < 2.0, however 50% of those demonstrate HER-2 amplification. Overall, 6.4% of HER-2 non-amplified cases with a ratio < 2.0 have an excess HER-2 copy number. In our full institutional pathology database, 11.2% of cases are HER2+, by either IHC or ISH. Lastly, 43% of cases overall, at UVM, from 2018 – March 2023 would fall into the current classification as HER-2 low (IHC 1+, or IHC 2+ and ISH negative), as well. Additionally, we have evaluated the change in IHC and ISH results that occurred with the changes in the ASCO/CAP guidelines in both 2013 and 2018, further data will be available for presentation. Conclusion: In this large single institutional analysis of dual HER-2 testing, only 12% of cases demonstrate HER-2 amplification, and overall in our institutional pathology database, from 2001 – March 2023, 11.2% of cases are HER-2+ by either IHC or ISH. In our dual tested cohort, only 13.6% of IHC 2+ cases demonstrate amplification, and 13.8% are non-amplified, but have excess HER-2 copy number. We demonstrate a numerical increase in the rate of amplification, and copy number in non-amplified cases, associated with IHC score. In all cases tested with IHC at UVM from 2018 – March 2023, we find 43% of cases meet the current criteria for HER-2 low. Finally, the ASCO/CAP guideline changes from 2013 to 2018 had modest impact on the overall percentage of HER-2+ cases in our series, further data on this impact will be available for presentation. Table. Simultaneously tested IHC and CISH data for new invasive breast cancer cases Citation Format: Xiaocao Xu, Abiy Ambaye, Kara Landry, Peter Kaufman. Dual HER2 Testing With Both Immunohistochemistry (IHC) and ISH (In Situ Hybridization): Long Term Analysis of Dual Testing in a Single Academic Institution [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-05.

Exo Ⅲ-assisted amplification signal strategy synergized with Au@Pt NFs/CoSe2 for sensitive detection of enrofloxacin

Overuse of enrofloxacin (ENR) has posed a potential threat to ecosystems and public health, so it is critical to sensitive and accurate determination of ENR residues. In this work, a novel ultra-sensitive and specific electrochemical aptasensor was fabricated based on the cobalt diselenide loaded gold and platinum nanoflowers (Au@Pt NFs/ CoSe2) and Exonuclease III (Exo III)-assisted cycle amplification strategy for the detection of ENR. Au@Pt NFs/ CoSe2 nanosheets as the substrate material, with large surface area, accelerate electron transfer and attach more DNA probes on the electrode substrate, have effectively enhanced the electrochemical performance of the electrode. With the existence of Enrofloxacin (ENR), the aptamer recognizes and binds to ENR, thus the signal probe cDNA was released and immobilized onto the electrode surface to hybridized with methylene blue (MB) labelled DNA (MB-DNA), thereby triggering the Exo III-assisted cycle for further signal amplification. As expected, the prepared aptasensor demonstrated excellent sensitivity and selectivity, with a wide linear range from 5.0 × 10−6 ng/mL to 1.0 × 10−2 ng/mL for ENR, a low detection limit of 1.59 × 10−6 ng/mL. Consequently, this strategy provided a promising avenue for ultrasensitive and accurate detection of ENR in milk samples.