Dual-labeled Probe Research Articles

A Dual-Labeled Multiplex Absolute Telomere Length Method to Measure Average Telomere Length

Background/Objectives: Telomeres consist of repetitive nucleotide sequences and associated proteins that safeguard chromosome ends from degradation and fusion with neighboring chromosomes. As cells divide, telomeres shorten due to the end-replication problem and oxidative stress, ultimately contributing to cellular senescence. Telomeres therefore play a role in cellular health and aging. Measuring telomere length has emerged as a significant biomarker in various fields of research, including aging, cancer, and chronic diseases. Accurate measurement of telomere length is critical for interpreting research findings and clinical applications. Variability in measurement techniques can lead to inconsistent results, underscoring the need for standardized protocols. Methods and Results: The Telomere Research Network (TRN), an initiative from the National Institute of Aging and National Institute of Environmental Health Sciences, has established recommended guidelines to standardize the measurement of telomere length using qPCR to ensure accuracy and reproducibility in population-based studies. The monochrome multiplex quantitative PCR (MMqPCR) assay has emerged as a robust method endorsed by the TRN for its accuracy and reproducibility in quantifying telomere length in epidemiology ad population based studies. The absolute telomere length (aTL) qPCR assay is currently being evaluated by the TRN for its capability to utilize an oligomer standard, enabling the generation of absolute telomere lengths. The oligomer feature facilitates a more direct comparison of results across experiments and laboratories. Conclusions: This paper outlines a novel dual-labeled multiplex aTL method by incorporating dual-labeled multiplex probes to measure average absolute telomere length, providing a clear advantage over the relative telomere length assay, which quantifies the ratio of telomeric repeats to single-copy gene numbers.

Simultaneous Genotyping of Three SNVs, rs5471, rs5472, and rs2000999 Involved in Serum Haptoglobin Levels by Fluorescent Probe-Based Melting Curve Analysis.

Haptoglobin (Hp) is a hemoglobin-binding acute-phase serum protein. Several single nucleotide variations (SNVs) within the Hp gene (HP) or Hp-related protein gene (HPR), such as rs5471 (A>C) and rs5472 (A>G) in HP promoter region and rs2000999 (G>A) in intron 2 of HRP, are suggested to correlate with the serum Hp levels. To determine these three SNVs simultaneously, a genotyping assay based on duplex dual-labeled fluorescent probes was developed. The method was then validated by analyzing genomic DNA from 121 Ghanaian and two Japanese subjects who had been previously genotyped for rs5471, rs5472, and rs2000999. Both rs5471 and rs5472 could be determined as haplotypes with a single FAM-labeled fluorescent probe, and rs2000999 could be genotyped with a HEX-labeled fluorescent probe. The results obtained with the present method were consistent with the previous results except for those of three Ghanaian subjects. All three subjects appear to have multiple HPR copy number variants characteristic of African populations, which may have led to incorrect results during previous genotyping. This method allows us to genotype these three SNVs in a relatively large number of samples, especially in African populations where rs5471 is uniquely distributed.

Quantitative BCR::ABL1 Assay Using Digital Real-Time PCR System: Clinical Applications and Insights

Background: Accurate quantification of BCR::ABL1 transcripts is paramount for the management of chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Despite advancements in real-time reverse transcription PCR (RT-qPCR), the need for more sensitive and precise assays remains. This study evaluated the diagnostic utility of digital reverse transcription PCR (dRT-PCR) for BCR::ABL1 quantification in assessing minimal residual disease (MRD). Material and methods. Twelve patients with CML or Ph+ ALL were included. Peripheral blood or bone marrow samples, collected in PaxGene RNA preservation tubes, were extracted with the automated QIASymphony SP instrument. dRT-PCR conducted on the LOAA analyzer, utilized the Dr. PCR BCR-ABL1 Detection Kit CE_IVD (OPTOLANE Technologies, South Korea). By employing a dual-labeled hydrolysis probe and target-specific primers, this protocol enabled the simultaneous amplification and detection of BCR::ABL1 transcripts e13a2 and e14a2 and ABL1. The resulting copy numbers/reactions were used to calculate the International Scale percentage (%IS) and the molecular reduction (MR) value and analyzed for clinical correlations. Results. Digital RT-PCR demonstrated high precision in quantifying BCR::ABL1 transcripts, the lowest %IS value being 0.02%. A consistent quality RNA sample totalizing 110 ng/reaction was crucial for successful reactions. This assay proved faster and easier than conventional RT-PCR and did not require standards or calibrators. Conclusion. This study supports the feasibility of digital RT-PCR for quantitative analysis. Future objectives for our laboratory include expanding our diagnostic repertoire using this platform, for enhancing patient care.

Abstract 2575: GD2 targeted dual-labeled intraoperative molecular imaging probe for neuroblastoma

Abstract Neuroblastoma (NB) is the most common pediatric extracranial solid tumor, with a 5-year survival rate under 50% for those with high-risk disease. Complete surgical resection is essential for these patients and is associated with improved outcomes. However, NB resection presents significant technical challenges due to encasement of vital structures that must be preserved, as well as remote deposits of disease. The current approach to find and safely resect tumor depends on the surgeon’s eyes and hands alone. Fluorescence tracers allow enhanced tumor visualization, but most are not molecularly targeted which leads to poor tumor to background signal. Further, fluorescent signal does not penetrate more than a few millimeters, a limitation that can be overcome by inclusion of a radiotracer. We, therefore, generated and evaluated a dual radio and fluorescent antibody-based probe targeting the GD2 tumor antigen to improve the identification of NB with widely available intraoperative handheld instruments. We generated an intraoperative probe (111In-αGD2-IRDye800CW) with retained affinity and stability. In vivo- Nu/j mice were injected with SK-N-BE(2) NB cells. After tumors grew for five weeks, mice were given tail vein injections of the probe. Gamma and optical biodistribution studies were performed to determine the specificity of binding and tumor to background signal four- and six-days post probe injection. Probe uptake was compared to an isotype. Gross dissections and surgeries aimed at improving completeness of resection were performed using a handheld fluorescent camera (SPY-PHI) and a gamma probe (Neoprobe). The handheld gamma probe detected xenografts with great sensitivity (average in vivo 818 cps of In111 in tumor region versus 34.8 cps in the tail). The fluorescent imaging showed high specific binding to the tumor region. Gamma biodistribution results indicated high accumulation within tumor (13.5 %ID/g) with lower accumulation in blood (3.49 %ID/g) and muscle (0.45 %ID/g). Uptake of the probe greatly exceeded isotype control antibody on days 4 and 6 post-agent injection, for both gamma and optical biodistributions. Initial tumor resection using 111In-DTPA-aGD2-IRDye800CW in an NB bearing mouse identified residual disease left behind when resection was performed using white light alone; the residual disease was then removed, guided by fluorescence imaging. Our dual-labeled probe demonstrated antigen specificity and allowed for the sensitive detection and visualization of NB in vivo with widely available intraoperative tools. This targeted probe could provide a means to improve surgical NB resection, by enabling localization of the all regions of tumor using radio-guidance, followed by clear definition of tumor margins with fluorescent signal, to maximize preservation of vital tissues. Ultimately, we believe this approach can improve safety, extent of NB resection, and patient outcomes. Citation Format: ReidAnn E. Sever, Lauren Taylor Rosenblum, Kayla C. Stanley, Dominic M. Menendez, W Barry Edwards, Marcus M. Malek, Gary Kohanbash. GD2 targeted dual-labeled intraoperative molecular imaging probe for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2575.

Lattice light-sheet DNA-PAINT microscopy with dual-labeled self-quenched imager probes

Lattice light-sheet DNA-PAINT microscopy with dual-labeled self-quenched imager probes

Dual-Labeled Single Fluorescent Probes for the Simultaneous Two-Color Visualization of Dual Organelles and for Monitoring Cell Autophagy.

Dual-labeled single fluorescent probes are powerful tools for studying autophagy on the molecular scale, yet their development has been hampered by design complexity and a lack of valid strategies. Herein, for the first time, we introduce a combinatorial regulation strategy to fabricate dual-labeled probes for studying autophagy by integrating the specific organelle-targeting group and the functional fluorescence switch into a pentacyclic pyrylium scaffold (latent dual-target scaffold). For proof of concept, we prepared a range of dual-labeled probes (TMOs) that display different emission colors in duple organelles. In these probes, TMO1 and TMO2 enabled the simultaneous two-color visualization of the lysosomes and mitochondria. The other probes (TMO3 and TMO4) discriminatively targeted lysosomes/nucleolus and lysosomes/lipid droplets (LDs) with dual-color emission characteristics, respectively. Intriguingly, by simply connecting the endoplasmic reticulum (ER) targeting group to the pentacyclic pyrylium scaffold, we created the first dual-labeled probe TMO5 for simultaneously labeling lysosomes/ER in distinctive fluorescent colors. Subsequently, using the dual-labeled probe TMO2, drug-induced mitophagy was successfully recorded by evaluating the alterations of multiple mitophagy-related parameters, and the mitophagy defects in a cellular model of Parkinson's disease (PD) were also revealed by simultaneous dual-color/dual-organelle imaging. Further, the probe TMO4 can track the movement of lysosomes and LDs in real time and monitor the dynamic process of lipophagy. Therefore, this work not only presents attractive dual-labeled probes to promote the study of organelle interactions during autophagy but also provides a promising combinatorial regulation strategy that may be generalized for designing other dual-labeled probes with multiple organelle combinations.

A dual-labeling fluorescent probe to track lysosomal polarity and endoplasmic reticulum dynamics during ferroptosis.

A polarity-sensitive probe was developed to simultaneously label lysosomes and endoplasmic reticulum (ER) via its dansylamide and rhodamine fluorescence, respectively, enabling ratiometric polarity detection and stable dual-labeling. The fragmented ER network and increased lysosomal polarity during ferroptosis were revealed, which facilitates the understanding of ferroptotic mechanisms.

Spatiotemporal Quantification of HER2-targeting Antibody-Drug Conjugate Bystander Activity and Enhancement of Solid Tumor Penetration.

Antibody-drug conjugate (ADC) has had a transformative effect on the treatment of many solid tumors, yet it remains unclear how ADCs exert bystander activity in the tumor microenvironment. Here, we directly visualized and spatiotemporally quantified the intratumor biodistribution and pharmacokinetics of different ADC components by developing dual-labeled fluorescent probes. Mechanistically, we found that tumor penetration of ADCs is distinctly affected by their ability to breach the binding site barrier (BSB) in perivascular regions of tumor vasculature, and bystander activity of ADC can only partially breach BSB. Furthermore, bystander activity of ADCs can work in synergy with coadministration of their parental antibodies, leading to fully bypassing BSBs and enhancing tumor penetration via a two-step process. These promising preclinical data allowed us to initiate a phase I/II clinical study of coadministration of RC48 and trastuzumab in patients with malignant stomach cancer to further evaluate this treatment strategy in humans.

Abstract PO-047: A novel saliva miRNA panel of promising diagnostic biomarkers for oral cancer: The association of miR-21 with smoking history

Abstract Introduction: Tobacco use is implicated in the carcinogenesis of oral squamous cell carcinoma (OSCC), which is associated with poor survival if not diagnosed early. It is urgent to develop a novel non-invasive and highly sensitive risk assessment and diagnostic method to screen OSCC. Here, we explored salivary miRNAs as a screening method for OSCC in a high-risk group of patients, such as smokers. Materials and methods: Saliva was collected from 44 individuals (23 HPV-negative OSCC; 21 controls; an equal number of smokers and non-smokers). Twenty head and neck cancer-related miRNA markers were analyzed by qPCR, using dual-labeled probes (miR-20A, miR-21, miR-27B, miR-29A, miR-29B, miR-29C, miR-31, miR-34a, miR-99a, miR-125a, miR-136, miR-139, miR-155, miR-192, miR-200A, miR-375, miR-425A, miR-451a, miR-504, miR-3928; RNU6 control), and by Welch’s t-test and ROC (receiver operating characteristic) curve; GraphPad Prism 7.0. Results: A panel of 4 miRNA markers (miR-21, miR-136, miR-3928, miR-29B) was found to be significantly overexpressed in the saliva of OSCC versus healthy controls with a diagnostic ability (p<0.05 by Welch’s t-test; AUC (area under the ROC curve): 64-85%, sensitivity: 50-67%, specificity: 32-38%; 95% confidence interval; by ROC curve analysis). “Oncomir” miR-21 levels (miR-21/RNU6) were found to be significantly higher in the saliva of OSCC patients with a smoking history (mean ± SD: 0.17 ± 0.19) versus never-smokers (mean ± SD: 0.0056 ± 0.0075) (p<0.05; by t-test) with a diagnostic ability (AUC: 90%, sensitivity: 68%, specificity: 30%; 95% confidence interval). Conclusions: We provide a novel panel of non-invasive, easy-to-apply, and sensitive biomarkers, for the diagnosis of oral cancer, including miR-21 in individuals with a smoking history, encouraging their validation in a large group of head and neck cancer patients. Citation Format: Dimitra Vageli, Panagiotis G. Doukas, Benjamin L. Judson. A novel saliva miRNA panel of promising diagnostic biomarkers for oral cancer: The association of miR-21 with smoking history [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-047.

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min.

Loop-mediated isothermal amplification (LAMP) is a commonly used alternative to PCR for point-of-care detection of nucleic acids due to its rapidity, sensitivity, specificity, and simpler instrumentation. While dual-labeled TaqMan probes are widely used in PCR for single-nucleotide polymorphism (SNP) genotyping, real-time LAMP primarily relies on turbidimetry or intercalator fluorescence measurements, which can be non-specific and generate false-positive results. In this study, we propose a closed-tube, dual-labeled RNA-modified probes and RNase H II-assisted real-time LAMP (RART-LAMP) method for SNP genotyping. Our findings indicate that (1) fluorescence signals were predominantly derived from probe hydrolysis rather than hybridization, (2) temperature-controlled hybridization between the probe and template ensured the specificity of SNP analysis, and (3) RNase H II hydrolysis between the target containing SNP sites and probes did not exhibit sequence specificity. Our RART-LAMP approach demonstrated excellent performance in genotyping C677T clinical samples, including gDNA extracted from blood, saliva, and swabs. More importantly, saliva and swab samples could be directly analyzed without any pretreatment, indicating promising prospects for nucleic acid analysis at the point of care in resource-limited settings.

Building a Teleost Fish Traceability Program Based on Genetic Data from Pacific Panama Fish Markets.

Fish tissue samples from 203 adult individuals were collected in the main ports and markets of the Pacific coast of Panama. Molecular identification based on a cytochrome oxidase I gene segment of all species was verified by GENBANK reference sequences. A total of 34 species from 14 families (Ariidae, Caranjidae, Centropomidae, Gerreidae, Haemulidae, Lobotidae, Lutjanidae, Malacanthidae, Mugilidae, Scianidae, Scombridae, Serranidae, Sphyraenidae, Stromateidae) were identified at the species level from 164 sequences. Additionally, three Caribbean species were molecularly identified among the analyzed samples (Mycteroperca xenarcha, Paralonchurus brasilensis and Lobotes surinamensis). Species diversity was slightly higher in the Gulf of Panama than in the Gulf of Chiriquí. For species with five or more individual sequences, genetic diversity and genetic connectivity parameters such as total number of haplotypes (H), haplotype diversity (Hd), and nucleotide diversity (π) were calculated. Overall, pelagic-migratory species showed higher values of genetic diversity than coastal and estuarine species with some exceptions. Connectivity between Gulf areas was compared using values of genetic distances and genetic differentiation (Fst). The high level of connectivity observed between the Gulf of Chiriqui and the Gulf of Montijo indicates the existence of a single stock in that area for the following species: Scomberomorus sierra, Caranx caninus and Lutjanus guttatus. The demographic history of the most common species was examined using Tajima's D values, suggesting population expansion for two snapper species, L. peru and L. argentiventris, having significant and higher values. Another important contribution from this research was the production of primers and dual-labeled probes for environmental DNA detection using qPCR for the five most abundant species (spotted rose snapper, yellow snapper, green jack, Pacific crevalle jack and the Pacific sierra fish). These markers represent a new set of tools for environmental DNA (eDNA) detection and molecular traceability of three commercially important fish species along the supply chain including landing sites and markets of the main fishery areas.

MALDI HiPLEX-IHC: multiomic and multimodal imaging of targeted intact proteins in tissues.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is one of the most widely used methods for imaging the spatial distribution of unlabeled small molecules such as metabolites, lipids and drugs in tissues. Recent progress has enabled many improvements including the ability to achieve single cell spatial resolution, 3D-tissue image reconstruction, and the precise identification of different isomeric and isobaric molecules. However, MALDI-MSI of high molecular weight intact proteins in biospecimens has thus far been difficult to achieve. Conventional methods normally require in situ proteolysis and peptide mass fingerprinting, have low spatial resolution, and typically detect only the most highly abundant proteins in an untargeted manner. In addition, MSI-based multiomic and multimodal workflows are needed which can image both small molecules and intact proteins from the same tissue. Such a capability can provide a more comprehensive understanding of the vast complexity of biological systems at the organ, tissue, and cellular levels of both normal and pathological function. A recently introduced top-down spatial imaging approach known as MALDI HiPLEX-IHC (MALDI-IHC for short) provides a basis for achieving this high-information content imaging of tissues and even individual cells. Based on novel photocleavable mass-tags conjugated to antibody probes, high-plex, multimodal and multiomic MALDI-based workflows have been developed to image both small molecules and intact proteins on the same tissue sample. Dual-labeled antibody probes enable multimodal mass spectrometry and fluorescent imaging of targeted intact proteins. A similar approach using the same photocleavable mass-tags can be applied to lectin and other probes. We detail here several examples of MALDI-IHC workflows designed to enable high-plex, multiomic and multimodal imaging of tissues at a spatial resolution as low as 5µm. This approach is compared to other existing high-plex methods such as imaging mass cytometry, MIBI-TOF, GeoMx and CODEX. Finally, future applications of MALDI-IHC are discussed.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

119 Single closed-tube qPCR assay with dual-labelled probes for improved sexing of equine embryos

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Fluorescence Melting Curve Analysis for Concurrent Genotyping of Three Tag SNPs in FUT3.

The synthesis of Lewis blood group antigens is governed by two fucosyltransferase genes, FUT2 and FUT3. Evidence is accumulating to suggest that functional polymorphisms of FUT2 and FUT3 are associated with a variety of clinical conditions. Fluorescence melting curve analysis (FMCA), using three different dual-labeled probes for concurrent genotyping of three single nucleotide polymorphisms (SNPs) of FUT3, c.59T>G, c.314C>T, and c.484G>A for Lewis-negative allele inference, was developed and validated using Ghanaian and Caucasian subjects. Although two other SNPs, c.55G>A, and c.61C>T, are located in the probe sequence for c.59T>G, it seems feasible to detect these two SNPs along with c.59T>G. The results obtained by probe-based FMCA were in perfect accordance with those obtained by Sanger sequencing for 106 Ghanaians and 100 Caucasians. The present method is useful and reliable for estimating Lewis-negative alleles on a relatively large scale.

A fluorogenic probe for core-fucosylated glycan-preferred ENGase

A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-β-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.

A dual-labeled fluorescent probe for visualization of dextranase activity in a simulated food digestion system

Molecular bioimaging of enzyme activity is rapidly emerging as a powerful strategy for accurate disease diagnostics. This work aims to prove that bioimaging of enzyme activity in food digestion with a fluorescent probe is feasible. In this study, a dual-labeled fluorescent probe with dextran-tetramethylrhodamine (TMR)-biotin conjugate (DTB) as the enzyme-cleavable unit, and biotin-(5-fluorescein) conjugate (FB) as the reference unit, was developed. It was immobilized in the agarose gel (the model food matrix) for the fluorescence quantification of dextranase activity. The probe manifested significantly ratiometric fluorescent signals (Igreen/Ired) in response to the enzyme-active reaction. Linear relationships of Igreen/Ired were obtained against the dextranase concentration ratio (C/C0). Igreen/Ired increased more rapidly with a greater dextranase diffusion rate, also supported by the more significant diffusion coefficient of fluorescently labeled dextranase in 0.5 wt% agarose gel (1.87 × 10-6 cm2 s−1). Our work provides more mechanistic evidence for enzyme activity imaging in food digestion.

Synthesis and characterization of N-rich fluorescent bio-dots as a reporter in the design of dual-labeled FRET probe for TaqMan PCR: A feasibility study.

DNA-based analytical techniques have provided an advantageous sensing assay in the realm of biotechnology. Bio-inspired fluorescent nanodots are a novel type of biological staining agents with excellent optical properties widely used for cellular imaging and diagnostics. In the present research, we successfully synthesized bio-dots with excellent optical properties and high-quantum yield from DNA sodium salt through the hydrothermal method. We conjugated the bio-dots with 3' Eclipse Dark Quencher (Eclipse)-labeled single-strand oligodeoxyribonucleotide according to carbodiimide chemistry, to design a fluorescence resonance energy transfer (FRET) probe. The results confirmed the prosperous synthesis and surface functionalization of the bio-dot. Analysis of size, zeta potential, and FTIR spectroscopy verified successful bioconjugation of the bio-dots with probes. UV-visibility analysis and fluorescence intensity profile of the bio-dot and bio-dot@probes represented a concentration-dependent quenching of fluorescent signal of bio-dot by Eclipse after probe conjugation. The results demonstrated that TaqMan PCR was not feasible using the designed bio-dot@probes. Our results indicated that bio-dot can be used as an efficient fluorescent tag in the design of fluorescently labeled oligonucleotides with high biocompatibility and optical features.

Abstract 2932: The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis

Abstract There are several molecular platforms routinely used to investigate genomic material. Quantitative polymerase chain reaction (qPCR) is ubiquitously used because of the ease of use and the precision however, it does not offer high multiplexing. While digital PCR (dPCR) is known for increased precision and sensitivity, most digital PCR platforms provide the same level of multiplexing as qPCR. Next-generation sequencing (NGS) is less precise than qPCR or dPCR and but has the highest multiplexing capability of the three technologies. Conventional digital PCR systems have been limited by the number of instruments required to conduct the workflow, time-to-result compared to qPCR, and level of multiplexing as compared to next-generation sequencing (NGS). Crystal Digital PCR™ and the naica system is a next-generation digital PCR platform that simplifies the number of instruments in the workflow, speeds up the time of results to less than 3 hours, and increases the number of detection colors to 6 spectrally distinct channels. By using a combination of dual-labelled fluorescent probes, multiplexed digital PCR provides a simple solution that makes it possible to use less of a precious sample when faced with limited quantity without sacrificing sensitivity. The naica system workflow utilizes microfluidic consumables, such as the Sapphire chip for high sensitivity applications, and the Opal chip for higher throughput studies. All reagents including primers and probes, master mix, and sample are inputted into the chips in one single loading step. Chips are transferred to the Geode instrument, where the sample is partitioned into a 2D-monolayer of droplet crystals and where all targets are amplified. After the PCR program has been completed, the same chips are then loaded into the Prism6 imager which detects all targets across 6 fluorescent channels and outputs an image of all droplets in a sample. Finally, Crystal Miner analysis software can be used to analyze and decipher the quantifications and expression of all targets in the assay. The 6-color naica system allows for high multiplex detection of targets previously unattainable by conventional digital PCR systems. With this advent, genomic scientists have an additional platform in the molecular toolbox that supports seamless assay development of oncology biomarker assays for gene expression, rare target detection, and liquid biopsy applications. Citation Format: Andrew Hoskins, Shekerah Primus, Mary Yazdani, Kimberley Gutierrez, Benjamin Foret, Fabien Bernard, Rémi Dangla. The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2932.