Low Background Signal Research Articles

A Novel Rhodamine B Derivative as a "Turn-on" Fluorescent Sensor for Cu2+ with High Selectivity and Sensitivity.

The number of "turn-on" fluorescent probes for Cu2+ is relatively limited, and interference from other metal cations presents a significant challenge for these sensors. In this study, we synthesized and characterized a rhodamine B-based sensor, designated as RBHP, using 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) and rhodamine B hydrazide. Selectivity, sensitivity, solvent effects, water content, and pH of RBHP in relation to Cu²⁺ were conducted. RBHP exhibited an exceptionally low fluorescence background signal in acetonitrile and demonstrated a "turn-on" fluorescent response to Cu²⁺. The PMBP-based acylhydrazone moiety and acetonitrile as the detection solvent are crucial for the selective detection. RBHP shows potential as a highly selective and sensitive fluorescent sensor for Cu2+.

Near-infrared light induced cathodic photoelectrochemical biosensor for duplex-specific nuclease mediated detection of miRNA166-a

The photoelectrochemical (PEC) biosensor is a new sensing platform which has the merits of low background signal, low cost, easy miniaturization and so on. However, at present, most PEC biosensors utilize visible light or ultraviolet light as the excitation light source. This type of light has high energy and may cause some light damage to the detected object and some photoelectric materials, thus limiting its application scope. Herein, based on NaYF4:Yb,Tm/BiOBr0.8I0.2 a cathode PEC biosensor that can be excited by near-infrared light was constructed to avoid the occurrence of anode false positives and light damage. Through two cycles of the miRNA cycle assisted by Duplex-Specific Nuclease (DSN) and the dissolved oxygen cycle assisted by gold nanoparticles during the detection process, the detection signal was amplified and miRNA166-a was sensitively detected. By using up-conversion materials, the near-infrared light of 980 nm can be converted into ultraviolet light and visible light, thus exciting the photogenerated carriers of BiOBr0.8I0.2. The photocurrent reaction shows a positive correlation with the quantity of gold nanoparticles present on the electrode surface, which are derived from the reporter molecules produced by the hybridization of miRNA166-a and H1. It has a good linear relationship and selectivity towards miRNA166-a, and the detection limit is 0.32 fM. This sensing method provides a cathode PEC biosensor, which can be excited by near-infrared light. The overall light damage of the system is low, and the possibility of false positive of anode PEC biosensor can be avoided, which has certain significance.

Ultrasensitive Electrochemical Biosensor with Powerful Triple Cascade Signal Amplification for Detection of MicroRNA.

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg2+-dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.

Recent advances in foodborne pathogen detection using photoelectrochemical biosensors: from photoactive material to sensing strategy

Rapid assessment and prevention of diseases caused by foodborne pathogens is one of the existing food safety regulatory issues faced by various countries, and it has received wide attention from all sectors of society. When the content of foodborne pathogens in food is higher than the limit standard and spreads in a certain way, it can cause disease outbreaks, which seriously threaten human health or life safety. Developing a novel method to detect foodborne pathogens accurately and rapidly is significant. Because of the limitations of complex steps, time-consuming, low sensitivity or poor selectivity of commonly used methods, a photoelectrochemical (PEC) biosensor based on electrochemistry is developed. Its advantages include a low background signal, fast response and simple operation. It also has broad application prospects for sensing, which has attracted wide attention. However, an organized summary of the latest PEC biosensors for foodborne pathogen sensing has not been reported. Therefore, this review introduces the recent advances in foodborne pathogen detection using PEC biosensors as follows: (i) the construction of PEC biosensors, (ii) the research status of PEC biosensors for the detection of foodborne pathogens and (iii) the direction of future development in this field. Hopefully, the study will provide some insight into developing more mature bio-sensing strategies to meet the practical needs of foodborne pathogen surveillance.

High-efficiency electrochemiluminescence of host-guest ligand-assembled gold nanoclusters preoxidated for ultrasensitive biosensing of protein

Herein, host–guest ligand-assembled gold nanoclusters (AuNCs) with highly efficient electrochemiluminescence (ECL) as an emitter and a programable tripedal deoxyribonucleic acid (DNA) walker with a well-regulated DNA track as the signal amplifier were used to develop an ultrasensitive biosensing platform for determining soluble urokinase-type plasminogen activator receptor (suPAR) related to chronic kidney disease. Notably, the host–guest ligand-assembled AuNCs with 6-aza-2-thiothymine (ATT) as the ligand and guanidine butyric acid (Gba) as the model (Gba/ATT-AuNCs) exhibited a strong and stable ECL signal under preoxidation, 25.6 times higher than that of traditional ATT-protected AuNCs (ATT-AuNCs). This is because of the dual-enhancement ECL mechanism of the suppression of nonradiative transitions by host–guest recognition and the improvement of the electrochemical redox rate by preoxidation. Furthermore, the trace target protein suPAR could be efficiently converted into a tripedal DNA walker to walk along a well-regulated square DNA orbit with high movement efficiency and low background signal, thereby considerably improving detection sensitivity. Thus, the established ECL sensing platform achieved the ultrasensitive detection of suPAR with a linear range from 10 fg/mL to 10 ng/mL and a limit of detection of 3.15 fg/mL, whose detection sensitivity was five orders of magnitude higher than that of the gold-standard enzyme-linked immunosorbent assay with the lowest detection concentration of 1 ng/mL. This strategy provides a novel enhanced ECL mode of AuNCs for constructing a sensitive biosensing platform and is expected to be extended to the trace detection of other protein markers in human body fluids for disease diagnosis.

A "Dual-Key-and-Lock" MRI Contrast Agent with T1-T2 Switchable Function for Accurate Diagnosis of Tumors.

Extremely small iron oxide nanoparticle (ESIONP)-based stimuli-responsive switchable MRI contrast agents (CAs) show great promise for accurate detection of tumors due to their outstanding advantages of high specificity and low background signal. However, currently developed ESIONP-based switchable CAs often suffer single-biomarker-induced responses, which lack absolute specificity to pathological tissues, potentially diminishing diagnostic accuracy. In this study, weak acidity and hypoxia, two of the most remarkable characteristics of tumors, are introduced as dual biomarker stimuli to construct an ESIONP-based switchable MRI CA (DKL-CA), with its signal switch controlled by a "dual-key-and-lock" strategy. Only when DKL-CA is exposed to a coexisting weakly acidic and hypoxic environment can monodispersed ESIONPs form nanoclusters, thereby realizing a switch from the T1 to T2 contrast. Moreover, DKL-CA exhibits favorable biosafety and the capacity for precise tumor diagnosis in tumor-bearing mice. Overall, DKL-CA paves the way for designing highly accurate ESIONP-based MRI CAs for tumor diagnosis.

High‐Efficiency Multilevel Phase Lenses with Nanostructures on Polyimide Membranes

AbstractThe emergence of planar meta‐lenses on flexible materials has profoundly impacted the long‐standing perception of diffractive optics. Despite their advantages, these lenses still face challenges in design and fabrication to obtain high focusing efficiency and resolving power. A nanofabrication technique is demonstrated based on photolithography and polyimide casting for realizing membrane‐based multilevel phase‐type Fresnel zone plates (FZPs) with high focusing efficiency. By employing advantageous techniques, these lenses with nanostructures are directly patterned into thin polyimide membranes. The computational and experimental results have indicated that the focusing efficiency of these nanostructures at the primary focus increases significantly with increasing the number of phase levels. Specifically, 16‐level phase lenses on a polyimide membrane can achieve a focusing efficiency of more than 91.6% of the input signal (9.5 times better than that of a conventional amplitude‐type FZP) and focus light into a diffraction‐limited spot together with very weak side‐lobes. Furthermore, these lenses exhibit considerably reduced unwanted diffraction orders and produce extremely low background signals. The potential impact of these lenses extends across various applications and techniques including microscopy, imaging, micro‐diffraction, remote sensing, and space flight instruments which require lightweight and flexible configurations.

Multifunctional Thermally Activated Delayed Fluorescence Carbon Dots for Temperature‐Responsive Sensor, Information Encryption, and Organelle Imaging

Abstract Thermally activated delay fluorescence (TADF) has great potential for information encryption, temperature detection, and bioimaging due to its long‐lived luminescence, temperature‐sensitive and high signal‐to‐noise ratio. However, it is still a challenge to establish TADF in aqueous environments. In this study, the composite with TADF (M‐FNCDs) is prepared using fluorine‐nitrogen co‐doped carbon dots (FNCDs) and melamine. It is worth mentioning that the M‐FNCDs show stable TADF under long‐wavelength excitation (470 nm) in aqueous environments. Moreover, the M‐FNCDs has distinctive temperature‐responsive properties and exhibit good linear relationships in the temperature range of 77–370 K. Simultaneously, M‐FNCDs suspension as the ink is utilized to realize information encryption/decryption due to their afterglow cannot be quenched in an aqueous solution. More importantly, M‐FNCDs with biocompatibility can target the mitochondria and lysosomes of living cells, and for the first time achieve the high signal‐to‐noise ratio and low background signal afterglow imaging of organelles. This work proposes a new strategy to prepare the stable TADF in aqueous solutions under long‐wavelength excitation and extend the TADF material potential applications in the future.

Fully Automated Cassette-Based Synthesis of 2-Deoxy-2-[18F]Fluorocellobiose Using Trasis AllInOne Module.

Due to the continuous rise in global incidence and severity of invasive fungal infections (IFIs), particularly among immunocompromised and immunodeficient patients, there is an urgent demand for swift and accurate fungal pathogen diagnosis. Therefore, the need for fungal-specific positron emission tomography (PET) imaging agents that can detect the infection in the early stages is increasing. Cellobiose, a disaccharide, is readily metabolized by fungal pathogens such as Aspergillus species. Recently, our group reported fluorine-18 labeled cellobiose, 2-deoxy-2-[18F]fluorocellobiose ([18F]FCB), for specific imaging of Aspergillus infection. The positive imaging findings with very low background signal on delayed imaging make this ligand a promising fungal-specific imaging ligand. Inspired by this result, the decision was made to automate the radiolabeling procedure for better reproducibility and to facilitate clinical translation. A Trasis AllInOne (Trasis AIO) automated module was used for this purpose. The reagent vials contain commercially available 2-deoxy-2-[18F]fluoroglucose ([18F]FDG), glucose-1-phosphate, and enzyme (cellobiose phosphorylase). A Sep-Pak cartridge was used to purify the tracer. The overall radiochemical yield was 50%-70% (n = 6, decay corrected) in 75-min synthesis time with a radiochemical purity of > 98%. This is a highly reliable protocol to produce current good manufacturing practice (cGMP)-compliant [18F]FCB for clinical PET imaging.

Dosimetry of [64Cu]FBP8: a fibrin-binding PET probe.

This study presents the biodistribution, clearance and dosimetry estimates of [64Cu]Fibrin Binding Probe #8 ([64Cu]FBP8) in healthy subjects. This prospective study included 8 healthy subjects to evaluate biodistribution, safety and dosimetry estimates of [64Cu]FBP8, a fibrin-binding positron emission tomography (PET) probe. All subjects underwent up to 3 sessions of PET/Magnetic Resonance Imaging (PET/MRI) 0-2 hours, 4h and 24h post injection. Dosimetry estimates were obtained using OLINDA 2.2 software. Subjects were injected with ~400 MBq of [64Cu]FBP8. Subjects did not experience adverse effects due to the injection of the probe. [64Cu]FBP8 PET images demonstrated fast blood clearance (half-life = 67 min) and renal excretion of the probe, showing low background signal across the body. The organs with the higher doses were: the urinary bladder (0.075 vs. 0.091 mGy/MBq for males and females, respectively); the kidneys (0.050 vs. 0.056 mGy/MBq respectively); and the liver (0.027 vs. 0.035 mGy/MBq respectively). The combined mean effective dose for males and females was 0.016 ± 0.0029 mSv/MBq, lower than the widely used [18F]fluorodeoxyglucose ([18F]FDG, 0.020mSv/MBq). This study demonstrates the following properties of the [64Cu]FBP8 probe: low dosimetry estimates; fast blood clearance and renal excretion; low background signal; and whole-body acquisition within 20 minutes in a single session. These properties provide the basis for [64Cu]FBP8 to be an excellent candidate for whole-body non-invasive imaging of fibrin, an important driver/feature in many cardiovascular, oncological and neurological conditions.

Conformational transformation of activator strand directly regulates CRISPR/Cas12a system activity for live cell sensing of multiple biomolecules

In recent years, biosensors designed based on the CRISPR/Cas12a system have opened up a new era in biosensing. However, the activator strand in the existing CRISPR/Cas12a-based sensing strategies are usually designed with 20 bases complementary to crRNA, it is difficult to completely shut down the trans-cleavage activity of the CRISPR/Cas12a system. To address this issue and simultaneously construct a biosensor with an “on-off-on” mode, we shortened the activator strand complementary to crRNA to 16 bases and embedded it into a hairpin structure depriving its ability to activate the Cas12a. We all know that the binding of aptamers with small molecules involves folding into a complex structure that is complementary to the surface of small molecules, thus forming a stable complex. To further expand the application of the CRISPR/Cas12a system in the field of biosensors, we selected small-molecule ATP and nucleolin as targets to confirm that the formation of the complex three-dimensional structure at the 3′- end of activator strand does not affect the activity of CRISPR/Cas12a system. Therefore, by designing only one allosteric activator strand, it is possible to regulate CRISPR/Cas12a system’s activity. This design not only reduces the number of DNA strands used in the sensing system, but also eliminates the need for complex concentration optimization operations. Furthermore, the stability of the intramolecular hairpin structure can minimize fluctuations in the background signal, ensuring stable and low-background signals in live cell applications, this further expands the toolbox of applications for the CRISPR/Cas12a system.

A novel NIR-II albumin-escaping probe for cerebral arteries and perfusion imaging in stroke mice model

In order to guide the formulation of post-stroke treatment strategy in time, it is necessary to have real-time feedback on collateral circulation and revascularization. Currently used near-infrared II (NIR-II) probes have inherent binding with endogenous albumin, resulting in significant background signals and uncontrollable pharmacokinetics. Therefore, the albumin-escaping properties of the new probe, IR-808AC, was designed, which achieved timely excretion and low background signal, enabling the short-term repeatable injection for visualization of cerebral vessels and perfusion. We further achieved continuous observation of changes in collateral vessels and perfusion during the 7-d period in middle cerebral artery occlusion mice using IR-808AC in vivo. Furthermore, using IR-808AC, we confirmed that remote ischemic conditioning could promote collateral vessels and perfusion. Finally, we evaluated the revascularization after thrombolysis on time in embolic stroke mice using IR-808AC. Overall, our study introduces a novel methodology for safe, non-invasive, and repeatable assessment of collateral circulation and revascularization in real-time that is crucial for the optimization of treatment strategies.

Catalytic hairpin assembly-based AIEgen/graphene oxide nanocomposite for fluorescence-enhanced and high-precision spatiotemporal imaging of microRNA in living cells

The low abundance, heterogeneous expression, and temporal changes of miRNA in different cellular locations pose significant challenges for both the detection sensitivity of miRNA liquid biopsy and intracellular imaging. In this work, we report an intelligently assembled biosensor based on catalytic hairpin assembly (CHA) and aggregation-induced emission (AIE), named as catalytic hairpin aggregation-induced emission (CHAIE), for the ultrasensitive detection and intracellular imaging of miRNA-155. To achieve such goal, tetraphenylethylene-N3 (TPE-N3) is used as AIE luminogen (AIEgen), while graphene oxide is introduced to quench the fluorescence. When the target miRNA is present, CHA reaction is triggered, causing the AIEgen to self-assemble with the hairpin DNA. This will restrict the intramolecular rotation of the AIEgen and produce a strong AIE fluorescence. Interestingly, CHAIE does not require any enzyme or expensive thermal cycling equipment, and therefore provides a rapid detection. Under optimal conditions, the proposed biosensor can determine miRNA in the concentration range from 2 pM to 200 nM within 30 min, with the detection limit of 0.42 pM. The proposed CHAIE biosensor in this work offers a low background signal and high sensitivity, making it applicable for highly precise spatiotemporal imaging of target miRNA in living cells.

Self-powered sandwich-type dual-mode sensor built on open bipolar electrode

Here, we developed a self-powered bipolar electrode (SP-BPE) based dual-mode sensor with electrochromic (EC) and photoelectrochemical (PEC) techniques for the visualized and sensitive detection of bisphenol A (BPA). An open BPE with the molecularly imprinted polymers (MIPs) modified electron injection area and the Prussian blue (PB) coated electrochromic area was fabricated on an indium tin oxide (ITO) glass. A kind of Zr-based metal-organic framework (UiO-66) coated TiO2 heterojunction (TiO2@UiO-66) was used for aptamer (Apt) immobilization and electron generation. BPA was selectively accumulated by MIPs, and subsequently recognized TiO2@UiO-66-Apt quantitatively, resulting in the formation of MIPs/BPA/Apt-TiO2@UiO-66 “sandwich” structure. Under UV-light, electrons were generated from the captured TiO2@UiO-66. For EC visualization measurements, electrons were injected into the PB film, causing a color change without an external power source. The extent of color change was proportional to the number of generated electrons, which correlated with the amount of captured BPA within a concentration range of 10−6–10−11 M. This allowed for the semi-quantification of BPA by naked eyes. For PEC detection, the BPE was used as the working electrode in a three-electrode system. The generated photocurrents signals could be digitally output for BPA with the concentrations ranging from 10−5 to 10−11 M. The SP-BPE sensors showed low background signal, improved selectivity and anti-interference abilities, and have demonstrated excellent performance in quantitative BPA analysis in water samples. They are particularly useful in scenarios where traditional power sources are limited or unavailable, and they offer opportunities for sustainable and environmentally friendly target detection.

Diagnostic Positron Emission Tomography Imaging with Zirconium-89 Desferrioxamine B Squaramide: From Bench to Bedside.

Molecular imaging with antibodies radiolabeled with positron-emitting radionuclides combines the affinity and selectivity of antibodies with the sensitivity of Positron Emission Tomography (PET). PET imaging allows the visualization and quantification of the biodistribution of the injected radiolabeled antibody, which can be used to characterize specific biological interactions in individual patients. This characterization can provide information about the engagement of the antibody with a molecular target such as receptors present in elevated levels in tumors as well as providing insight into the distribution and clearance of the antibody. Potential applications of clinical PET with radiolabeled antibodies include identifying patients for targeted therapies, characterization of heterogeneous disease, and monitoring treatment response.Antibodies often take several days to clear from the blood pool and localize in tumors, so PET imaging with radiolabeled antibodies requires the use of a radionuclide with a similar radioactive half-life. Zirconium-89 is a positron-emitting radionuclide that has a radioactive half-life of 78 h and relatively low positron emission energy that is well suited to radiolabeling antibodies. It is essential that the zirconium-89 radionuclide be attached to the antibody through chemistry that provides an agent that is stable in vivo with respect to the dissociation of the radionuclide without compromising the biological activity of the antibody.This Account focuses on our research using a simple derivative of the bacterial siderophore desferrioxamine (DFO) with a squaramide ester functional group, DFO-squaramide (DFOSq), to link the chelator to antibodies. In our work, we produce conjugates with an average ∼4 chelators per antibody, and this does not compromise the binding of the antibody to the target. The resulting antibody conjugates of DFOSq are stable and can be easily radiolabeled with zirconium-89 in high radiochemical yields and purity. Automated methods for the radiolabeling of DFOSq-antibody conjugates have been developed to support multicenter clinical trials. Evaluation of several DFOSq conjugates with antibodies and low molecular weight targeting agents in tumor mouse models gave PET images with high tumor uptake and low background. The promising preclinical results supported the translation of this chemistry to human clinical trials using two different radiolabeled antibodies. The potential clinical impact of these ongoing clinical trials is discussed.The use of DFOSq to radiolabel relatively low molecular weight targeting molecules, peptides, and peptide mimetics is also presented. Low molecular weight molecules typically clear the blood pool and accumulate in target tissue more rapidly than antibodies, so they are usually radiolabeled with positron-emitting radionuclides with shorter radioactive half-lives such as fluorine-18 (t1/2 ∼ 110 min) or gallium-68 (t1/2 ∼ 68 min). Radiolabeling peptides and peptide mimetics with zirconium-89, with its longer radioactive half-life (t1/2 = 78 h), could facilitate the centralized manufacture and distribution of radiolabeled tracers. In addition, the ability to image patients at later time points with zirconium-89 based agents (e.g. 4-24 h after injection) may also allow the delineation of small or low-uptake disease sites as the delayed imaging results in increased clearance of the tracer from nontarget tissue and lower background signal.

Low-background fluorescent biosensor based on graphene oxide and aptamer biorecognition for sensitive detection of kanamycin

The detection of kanamycin has attracted extensive attention in the field of food safety. A label-free, low-background fluorescent biosensor was successfully constructed based on graphene oxide and aptamer biorecognition in this paper for kanamycin detection in milk samples. In this biosensor, the integrated probe with the aptamer sequence of kanamycin and the G-rich sequence was designed. The aptamer sequence of kanamycin was used as the recognition part, the G-quadruplex/N-methyl mesoporphyrin IX (NMM) complex as the fluorescent probe, and graphene oxide as the fluorescent quencher. Initially, the integrated probe could be adsorbed on the graphene oxide surface through π-π stacking interactions, while the fluorescence of NMM could be efficiently quenched because of the resonance energy transfer between NMM and graphene oxide, which was beneficial to the low background signal of the system. During the detection process, the aptamer sequence preferred to bind to kanamycin, forming aptamer-kanamycin complex. At the same time, the G-rich sequence folded into G-quadruplex structure that specifically bound to NMM under the action of K+, forming G-quadruplex/NMM complex. Under the synergistic effects of the two complexes, the integrated probe with aptamer-kanamycin complex and G-quadruplex/NMM complex was released from the graphene oxide surface due to the steric hindrance effect, restoring the strong fluorescence signal. By combining graphene oxide and G-quadruplex/NMM complex, the fluorescent biosensor could effectively achieve label-free and low-background detection of kanamycin. The linear calibration curve was in the range of 1–400 nM with a detection limit of 0.60 nM for kanamycin. It was successfully applied to analyze raw milk samples (spiked kanamycin) with recoveries of 98.33–101.25 % and also exhibited high selectivity for kanamycin. The experimental data showed that the biosensor was a potentially powerful tool for monitoring food safety.

UIO66 low background signal and fluorescence synergism strategy for highly sensitive detection of Salmonella typhimurium

Successful construction of a detection method for Salmonella typhimurium (S. typhimurium) based on the synergy of hybridization chain reaction (HCR) and fluorescence was realized in this paper. First, the aptamer modified with the quenching group Black Hole Quencher-1 acid (BHQ1) was immobilized on the magnetic beads in combination with the complementary chain of the aptamer modified with 6-carboxyfluorescein (6-FAM). Second, S. typhimurium and cDNA-6-FAM immobilized on magnetic beads competitively bound to the aptamer. Finally, the cDNA-6-FAM was released after magnetic separation acted as a promoter to trigger HCR amplification when the target presented. The fluorescence signal could be significantly improved by the combination of green SYBR Green I (SGI) and HCR long double-stranded DNA and the fluorescent synergy of 6-FAM and SGI. Because of the separation of target and its aptamer, the trigger strand was abstracted by magnetic separation. There was no HCR to generate long double-stranded DNA, and the fluorescence of excess hairpin/SGI could be adsorbed through UIO66 so that only a very low background signal was detected. This fluorescent sensor was capable of monitoring S. typhimurium in the range of 10–3.2 × 107 CFU mL−1 with a limit of detection as low as 1.5 CFU mL−1. Because of the excellent properties of the aptasensor and the validity of SGI fluorescence synergy, this HCR enzyme-free amplification strategy could be generalized to other areas.

Plasmonic colorimetry and G-quadruplex fluorescence-based aptasensor: A dual-mode, protein-free and label-free detection for OTA

G-quadruplexes (G4) have received significant attention in the field of aptasensors owing to their unique physicochemical characteristics. A dual-mode, protein-free and label-free aptamer sensor based on plasmonic colorimetry and G4 fluorescence (PC@GF-aptasensor) was proposed for ochratoxin A (OTA). Colorimetry mode was achieved through the surface plasmon resonance (SPR) effect, which related to the OTA-Apt-based G4–OTA. The fluorescence mode was reflected by the insertion of thioflavin T (ThT) into G4–OTA. The OTA could be interpreted via three readouts: (1) naked eye (LOD of 2.0 ng mL−1), (2) smartphone (LOD of 1.65 ng mL−1), and (3) spectrofluorometer (LOD of 0.93 ng mL−1). The PC@GF-aptasensor exhibited several advantages, such as a standardised recognition group, simplified operation, low background signal, and practicality. The proposed PC@GF-aptasensor integrated SPR-based multicolour interpretation and ThT-inserted fluorescence reflection to obtain a dual-mode optical biosensor, which may provide valuable insights for the development of other targets with G4-based aptamers.

Colorimetric biosensor based on aptamer recognition-induced multi-DNA release and peroxidase-mimicking three-way junction DNA-Ag/PtNCs for the detection of Salmonella typhimurium

Salmonella typhimurium, as a major foodborne pathogen, poses a serious threat to public health safety worldwide. Here, we present a colorimetric biosensor based on aptamer recognition-induced multi-DNA release and peroxidase-mimicking three-way junction DNA-silver/platinum bimetallic nanoclusters (3WJ/DNA-Ag/PtNCs) for the detection of S. typhimurium. In this method, S. typhimurium specifically binds to the aptamer and releases multiple cDNAs to form the three-way junction DNA structure and synthesize silver/platinum bimetallic nanoclusters, which induces signaling changes. Interestingly and importantly, the use of 3WJ/DNA as the template for synthesizing Ag/PtNCs gives the method an extremely low background signal. Under the optimal conditions, the constructed biosensor had a linear response range of 2.6 × 102-2.6 × 106 CFU/mL and a detection limit of 2.6 × 102 CFU/mL for the detection of S. typhimurium. In addition, the proposed method can effectively detect S. typhimurium in milk.

Abstract 2424: Detection of prostate cancer in urine by fragment-level DNA methylation analysis

Abstract Introduction: Screening for prostate cancer (PCa) with molecular markers is hindered by low tumor allele fraction (TAF) in liquid biopsy samples such as blood or urine. Others have suggested that tumor allele enrichment can be achieved by evaluating fragment-level methylation patterns generated by next generation sequencing (NGS). Here, we test this concept using target capture NGS on cfDNA from prediagnostic collected urine samples to discriminate PCa from otherwise healthy men. Methods: Buffered urines were collected from 23 patients with biopsy confirmed PCa (Gleason score 6-9) and 24 otherwise healthy age matched cancer-free controls. cfDNA was isolated and enzymatic methyl-seq libraries were prepared and enriched by hybrid capture using probes mapping to 197 differentially methylated regions (DMRs) from a previous PCa/normal tissue marker discovery study in our lab. These DMRs were subsequently vetted independently using a methylome-wide dataset from a cohort of 36 clean and disease control urines to ensure low non-cancer background signal (<1%). Indexed libraries were sequenced at 560X deduplicated coverage and fragment-level CpG methylation information was extracted from individual sample .bam files. Mapped sequencing reads exhibiting concordant methylation were used to derive a quantitative α-score. Reads had to contain at minimum 5 CpGs and have an α-value (% 5mC per read) > 60%. Positivity for the 197 markers required an α-score greater than 6 sigma (σ) above the mean of the control arm. Values were variance-adjusted to ensure equal weighting. Beta (β) analysis (mean % 5mC across all reads) of the data was also performed for comparison. Results: Of the 197 markers, 187 were positive for one or more PCa patients. Tumor-derived fragments were detected in 22 of 23 cases, which included 8 G-6, 9 G-7, 2 G-8, and 3 G-9 patients. 1 G-7 was missed, however the sequencing coverage for this sample was significantly lower than the others due to poor library enrichment. 2 of the 24 healthy controls were positive, one with extremely high fragment counts. Upon re-review of the full control cohort, this patient had been diagnosed with bladder cancer 2 years after urine collection. The 2nd patient had was diagnosed with lung cancer in a similar timeframe. The other 22 confirmed cancer-free controls had α-scores below the 6σ threshold. Using a co-captured reference gene (B3GALT6) to mimic genomic equivalents, the TAFs ranged from 0.015% to 8%. β analysis of the data detected only 13 PCa patients (>6σ) and marker positivity was lower than the fragment-level approach. Conclusion: Target capture NGS coupled with fragment-level analysis is a rational approach for sensitive and specific detection of low tumor-derived DNA in liquid biopsy samples. For PCa assay in urine, we were able to uncover positivity in cancer patients approaching 1 in 10,000 ctDNA dilution levels. These results warrant further validation studies with this and other cancers. Citation Format: Tibor Bedekovics, William R. Taylor, Zhifu Sun, Saurabh Baheti, Xiaoming Cao, Kelli N. Burger, Karen A. Doering, Patrick H. Foote, Benjamin Gochanour, Douglas, W. Mahoney, Matthew T. Gettman, John B. Kisiel. Detection of prostate cancer in urine by fragment-level DNA methylation analysis [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 2424.