Probe Design Research Articles

Highly efficient removal and real-time visual detection of fluoride ions using ratiometric CAU-10-NH2@RhB: Probe design, sensing performance, and practical applications

The extensive use of fluoride in agriculture, industry, medicine, and daily necessities has raised growing concerns about fluoride residue. To date, real-time visual detection and efficient removal of fluoride ions from water remain greatly desirable. Herein, nano-CAU-10-NH2@RhB is introduced as a ratiometric fluorescent probe and efficient scavenger for the intelligent detection and removal of fluoride ions. CAU-10-NH2@RhB is readily obtained through one-pot synthesis and exhibits high sensitivity and selectivity for real-time fluoride ion detection, with a naked-eye distinguishable color change from pink to blue. A portable device for point-of-care testing was developed based on color hue analysis readout using a smartphone. A quantitative response was achieved across a wide concentration range, with a detection limit of 54.2 nM. Adsorption experiments suggest that nano-CAU-10-NH2@RhB serves as an efficient fluoride ion scavenger, with a fluoride adsorption capacity of 49.3 mg/g. Moreover, the mechanistic study revealed that hydrogen bonds formed between fluoride ions and amino groups of CAU-10-NH2@RhB are crucial for the detection and adsorption of fluoride ions. This analysis platform was also used for point-of-care quantitative visual detection of fluoride ions in food, water, and toothpaste.

Unraveling the Molecular Mechanisms by Which the miR171b-SCL6 Module Regulates Maturation in Lilium.

Lilium is one of the most widely cultivated ornamental bulbous plants in the world. Although research has shown that variable temperature treatments can accelerate the development process from vegetative to reproductive growth in Lilium, the molecular regulation mechanisms of this development are not clear. In this study, Lbr-miR171b and its target gene, LbrSCL6, were selected and validated using transgenic functional verification, subcellular localization, and transcriptional activation. This study also investigated the differential expression of Lbr-miR171b and LbrSCL6 in two temperature treatment groups (25 °C and 15 °C). Lbr-miR171b expression significantly increased after the temperature change, whereas that of LbrSCL6 exhibited the opposite trend. Through in situ hybridization experiments facilitated by the design of hybridization probes targeting LbrSCL6, a reduction in LbrSCL6 expression was detected following variable temperature treatment at 15 °C. The transgenic overexpression of Lbr-miR171b in plants promoted the phase transition, while LbrSCL6 overexpression induced a delay in the phase transition. In addition, LbrWOX4 interacted with LbrSCL6 in yeast two-hybrid and bimolecular fluorescence complementation assays. In conclusion, these results explain the molecular regulatory mechanisms governing the phase transition in Lilium.

Self-stacked small molecules for ultrasensitive, substrate-free Raman imaging in vivo.

Raman spectroscopy using surface-enhanced Raman scattering (SERS) nanoprobes represents an ultrasensitive and high-precision technique for in vivo imaging. Clinical translation of SERS nanoprobes has been hampered by biosafety concerns about the metal substrates used to enhance Raman signals. We report a set of small molecules with bis-thienyl-substituted benzobisthiadiazole structures that enhance Raman signal through self-stacking rather than external substrates. In our technique, called stacking-induced charge transfer-enhanced Raman scattering (SICTERS), the self-stacked small molecules form an ordered spatial arrangement that enables three-dimensional charge transfer between neighboring molecules. The Raman scattering cross-section of SICTERS nanoprobes is 1350 times higher than that of conventional SERS gold nanoprobes of similar particle size. SICTERS outperforms SERS in terms of in vivo imaging sensitivity, resolution and depth. SICTERS is capable of noninvasive Raman imaging of blood and lymphatic vasculatures, which has not been achieved by SERS. SICTERS represents an alternative technique to enhance Raman scattering for guiding the design of ultrasensitive substrate-free Raman imaging probes.

The effect of teacher education supported through consultation on acquisition communication skills in children with autism spectrum disorder

The purpose of this paper is to measure the effects of the consultation method that includes teaching and practicing the mand-modelling technique to teachers and parents to improve the communication skills of children with autism. Multiple probe design, which is one of the single-case research designs, was used in the study. The findings of this research show that families with children with autism can be successful in improving their children’s limited verbal communication skills through receiving training in milieu teaching techniques that parents receive from their children’s teachers and perform in parallel with their teachers. The results showed that using the consultation method to teach the mand-modelling technique to special education teachers by ensuring that the family participates in the education is effective in developing pragmatic language in children with autism.

Reversible Fluorescent Probes for Dynamic Imaging of Liver Ischemia-Reperfusion Injury.

ConspectusHepatic ischemia-reperfusion injury (HIRI) is an inevitable complication of clinical surgeries such as liver resection or transplantation, often resulting in postoperative liver dysfunction, hepatic failure in up to 13% of postresection patients, and early graft failure in 11-18% of liver transplantation patients. HIRI involves a series of biochemical events triggered by abnormal alterations in multiple biomarkers, characterized by short lifespans, dynamic changes, subcellular regional distribution, and multicollaborative regulation. However, traditional diagnosis, including serology, imaging, and liver puncture biopsy, suffers from low sensitivity, poor resolution, and hysteresis, which hinder effective monitoring of HIRI markers. Thus, to address the unique properties of HIRI markers, there is a pressing demand for developing novel detection strategies that are highly selective, transiently responsive, dynamically reversible, subcellular organelle-targeted, and capable of simultaneous multicomponent analysis.Optical probe-based fluorescence imaging is a powerful tool for real-time monitoring of biomarkers with the advantages of high sensitivity, noninvasiveness, rapid analysis, and high-fidelity acquisition of spatiotemporal information on signaling molecules compared with conventional methods. Moreover, with the growing demand for continuous monitoring of biomarkers, probes with reversible detection features are receiving more and more attention. Importantly, reversible probes can not only monitor fluctuations in marker concentrations but also distinguish between transient bursts of markers during physiological events and long-term sustained increases in pathological marker levels. This can effectively avoid false-positive test results, and in addition, reversible probes can be reutilized with green and economical features. Therefore, our team has employed various effective methods to design reversible optical probes for HIRI. We proposed reversible recognition strategies based on specific reactions or interactions to detect dynamic changes in markers. Given the biomarkers' unique signaling in subcellular organelles and the synergistic regulatory properties of multiple markers for HIRI, bifunctional reversible detection strategies are exploited, including organelle-targeted reversible and multicomponent simultaneous detection. With these strategies, we have tailored a variety of high-fidelity fluorescent probes for a series of HIRI markers, including reactive oxygen/nitrogen species (O2•- and ONOO-), ATP, protein (Keap1), mitochondrial DNA, etc. Utilizing the probes, the in situ dynamic imaging detection of the HIRI markers was successfully achieved. While performing the precise examination of the earlier occurrence of HIRI disease and visualizing the real-time monitoring of the disease process, we have also further elucidated the HIRI-associated signaling pathways. It is envisioned that our summarized work will inspire the design of future reversible fluorescent probes and help to improve the clinical diagnosis and therapeutic efficiency of these diseases.

Eddy Current Sensor Probe Design for Subsurface Defect Detection in Additive Manufacturing.

Pore and crack formation in parts produced by additive manufacturing (AM) processes, such as laser powder bed fusion, is one of the issues associated with AM technology. Surface and subsurface cracks and pores are induced during the printing process, undermining the printed part durability. In-situ detection of defects will enable the real-time or intermittent control of the process, resulting in higher product quality. In this paper, a new eddy current-based probe design is proposed to detect these defects in parts with various defects that mimic pores and cracks in additively manufactured parts. Electromagnetic finite element analyses were carried out to optimize the probe geometry, followed by fabricating a prototype. Artificial defects were seeded in stainless steel plates to assess the feasibility of detecting various flaws with different widths and lengths. The smallest defect detected had a 0.17 mm radius for blind holes and a 0.43 mm notch with a 5 mm length. All the defects were 0.5 mm from the surface, and the probe was placed on the back surface of the defects. The surface roughness of the tested samples was less than 2 µm. The results show promise for detecting defects, indicating a potential application in AM.

Effectiveness of the interaction unit method in teaching addition and subtraction problem-solving skills to students with intellectual disability

This study examines the effectiveness of the interaction unit method, which is presented virtually, in teaching addition and subtraction problem-solving skills to students with intellectual disability (ID). The research was conducted with three students (one girl and two boys) aged 15–16 years who were diagnosed with ID and were attending a special education vocational school in Istanbul. Multiple probe design with probe trials across the subjects was used. The dependent variables include the participants’ level of solving addition and subtraction problems, and the independent variable is the teaching via the virtual interaction unit method (VIUM). We found that by using this method, students with ID could acquire the skills to solve addition and subtraction problems. Moreover, it was determined that the students continued to maintain these skills one and two weeks after the intervention and were able to generalize different environments, tools, and people. Considering the social validity of the research obtained from teachers’ opinions, it was concluded that the opinions about the VIUM were positive.

Evaluation of prostate cancer detection using micro-ultrasound versus MRI through co-registration to whole-mount pathology

Micro-ultrasound has recently been introduced as a low-cost alternative to multi-parametric MRI for imaging prostate cancer. Early clinical studies have demonstrated promising results; however, robust validation via comparison with whole-mount pathology has yet to be achieved. Due to micro-ultrasound probe design and tissue deformation during scanning, it is difficult to accurately correlate micro-ultrasound imaging planes with ground truth whole-mount pathology slides. In this study, we developed a multi-step methodology to co-register micro-ultrasound and MRI to whole-mount pathology. The three-step process had a registration error of 3.90 ± 0.11 mm and consists of: (1) micro-ultrasound image reconstruction, (2) 3D landmark registration of micro-ultrasound to MRI, and (3) 2D capsule registration of MRI to whole-mount pathology. This process was then used in a preliminary reader study to compare the diagnostic accuracy of micro-ultrasound and MRI in 15 patients who underwent radical prostatectomy for prostate cancer. Micro-ultrasound was found to have equivalent performance to retrospective MRI review for index lesion detection (91.7% vs. 80%), while demonstrating an increased detection of tumor extent (52.5% vs. 36.7%) with similar false positive regions-of-interest (38.3% vs. 40.8%). Prospective MRI review had reduced detection of index lesions (73.3%) and tumor extent (18.9%) but improved false positive regions-of-interest (22.7%) relative to micro-ultrasound and retrospective MRI. Further evaluation is needed with a larger sample size.

Optimal Probe Design for Fluorescence Resonance Energy Transfer–PCR: Simultaneous Detection and Differentiation of Similar Targets

Fluorescence resonance energy transfer (FRET)-PCR is widely recognized for its high sensitivity and specificity in pathogen detection. However, there are some gaps in probe design when it is applied for simultaneous detection and differentiation of similar targets. This study aims to investigate the effects of the numbers and position of nucleotide mismatches (NM) in probe on PCR efficiency and melting temperature (Tm). The results indicated that NM at the center reduces amplification efficiency and Tm more significantly than NM at the 5′-terminal or 3′-terminal of the probe.

Mechanism and design of organic afterglow luminescent probes for cancer theranostics

Organic afterglow luminescent probes (OALPs), characterized by their long-lasting luminescence after irradiation (by light, ultrasound, or X-rays) cessation, are pivotal tools in autofluorescence-free optical imaging. They exhibit ultra-low background noise interference, enhancing imaging sensitivity and ensuring clearer, more reliable imaging results. Moreover, they offer deeper tissue penetration compared to traditional optical imaging modalities, providing various information from deep tissues. Recently developed sonoafterglow and radioafterglow further enhance tissue penetration depth. This review outlines 2 design approaches for OALPs: coencapsulation and conjugation, which are derived from their luminescent mechanism. Guided by these strategies, researchers have designed 3 types of OALPs: near-infrared OALPs, responsive OALPs, and ratiometric OALPs. Additionally, we also provided examples of how OALPs are integrated with therapy and applied in the field of cancer theranostics. Finally, we discuss certain challenges encountered in the advancement of the next generation of OALPs, aiming to broaden their scope of applications.

Towards clinical application of freehand optical ultrasound imaging

Freehand optical ultrasound (OpUS) imaging is an emerging ultrasound imaging paradigm that uses an array of fibre-optic, photoacoustic ultrasound sources and a single fibre-optic ultrasound detector to perform ultrasound imaging without the need for electrical components in the probe head. Previous freehand OpUS devices have demonstrated capability for real-time, video-rate imaging of clinically relevant targets, but have been hampered by poor ultrasound penetration, significant imaging artefacts and low frame rates, and their designs limited their clinical applicability. In this work we present a novel freehand OpUS imaging platform, including a fully mobile and compact acquisition console and an improved probe design. The novel freehand OpUS probe presented utilises optical waveguides to shape the generated ultrasound fields for improved ultrasound penetration depths, an extended fibre-optic bundle to improve system versatility and an overall ruggedised design with protective elements to improve probe handling and protect the internal optical components. This probe is demonstrated with phantoms and the first multi-participant in vivo imaging study conducted with freehand OpUS imaging probes, this represents several significant steps towards the clinical translation of freehand OpUS imaging.

Measuring Integrin Force Loading Rates Using a Two-Step DNA Tension Sensor.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (piconewton) integrin-ECM forces have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrin receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop an LR probe composed of an engineered DNA structure that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN (hairpin unfolding) and a high force threshold at 47 pN (duplex shearing). These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live cells. Automated analysis of tens of thousands of events from eight cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 s. A subset of these events mature in magnitude to >47 pN with a median loading rate of 1.1 pN s-1 and primarily localize at the periphery of the cell-substrate junction. The LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of molecular mechanotransduction in living systems.

Online Intervention to Prevent Summer Learning Loss For Struggling First Grade Writers

During summer vacation, lower-achieving students experience learning loss in writing. This study investigated online summer tutoring for first grade writers. The four participants were six months to two years below grade level on at least two of written expression, spelling, and handwriting fluency. A multiple probe design with five phases was used: (1) Baseline; (2) transcription intervention; (3) mid-assessment; (4) Self-Regulated Strategy Development intervention (SRSD); and (5) post-assessment. The transcription phase increased handwriting fluency, spelling in text, and spelling of frequently misspelled words, but not handwriting legibility. The SRSD intervention produced large gains in word count, holistic text quality, and Test of Early Written Language-3 Contextual Writing. Online tutoring can prevent summer learning loss and improve written expression.

Gingival phenotype classification by visual and probe visibility assessments: Relationship with thickness and probe design.

This study investigated the agreement among dentists in classifying gingival phenotype (GP) through periodontal probe visibility (PPV) assessment with various probe types and the visual method. Additionally, the relationship between GP classifications and gingival thickness (GT) was evaluated. Photographs were taken with standard periodontal probe (SPP), color-coded periodontal probe (CCPP) tips in white, green, and blue, as well as metal phenotype probe (MPP) tips in gray and black. Evaluators (periodontist, periodontics resident, endodontics resident, dental student) assessed the photographs and classified the GPs. GT was measured by trans gingival probing. Visual method showed poor to fair agreement to classify GP. The lowest agreement regarding PPV was noted with white-tipped CCPP. The highest agreement in singular PPV was observed with CCPP blue (κ=0.932), followed by CCPP green (κ=0.791), MPP black (κ=0.783), SPP (κ=0.730), and MPP gray (κ=0.690). Combined PPV data revealed fair to moderate agreement with CCPP and moderate to substantial agreement with MPP in GP classification. The corresponding GT to different GP classifications based on combined PPV were comparable. The agreement between SPP and CCPP in classifying non-thin phenotypes was 89.8%, while the agreement between SPP and MPP was 75.4%. Based on PPV, no significant GT cutoff value was found to distinguish between thin and non-thin phenotypes. Determining a precise GT that guarantees the visibility of a given probe can be difficult when evaluating GP. Regardless of the type of probe, the PPV method has a high potential for misclassifying GP, despite having an acceptable agreement. Gingival phenotype (GP) is constituted by thickness of the gums and width of keratinized tissue around teeth. Direct visual evaluation or evaluating a periodontal probe's visibility beneath gums are established techniques to classify gingival phenotype. This study investigated how dentists classify GP using visual assessments and different types of periodontal probes, while also exploring the relationship between GP classifications and gingival thickness. Results showed varied agreement among dentists in classifying GP, with lower agreement observed when using certain types of probes, notably the white-tipped phenotype probe. The highest agreement was found with the blue phenotype probe. Data from periodontal probe visibility assessments indicated fair to moderate agreement with certain probes, suggesting some inconsistency in classification methods. Interestingly, GP classification with visual assessments or probes did not correlate with gingival thickness, which may highlight the importance of considering both factors in clinical practice. These findings underline the need for attention when relying solely on visual assessments or specific probe types for accurate GP classification.

Effect of Experimental Parameters on the Electrochemical Current during Scanning Electrochemical Microscopy of Living Cells

Scanning Electrochemical Microscopy (SECM) is gaining popularity for its application to live cell imaging to monitor electrochemical processes occurring at the interface of living cells and electrodes. [1] Although many studies have been reported varying the instrumental parameters and using various probe designs, [2] the influence of experimental parameters on the electrochemical current must not be neglected, in order to generate reliable results. Our work explores parameters, such as temperature, light exposure and cell media during the SECM analysis of Adenocarcinoma Cervical cancer (HeLa) cells. The experimental and numerical studies with temperature variation revealed that temperature changes of 2ᵒC influence the reaction kinetics of single living cells. [3] Further, it was observed during light exposure that the electrochemical current becomes stabilized after 20 minutes of constant light environment compared to dark, where the standard deviation of the electrochemical current showed an increase in trend overtime. Furthermore, electrochemical current responses of HeLa cells in Phosphate Buffered Solution (PBS), Dubelcco’s Modified Eagle Medium (DMEM) and DMEM with 10% Fetal Bovine Serum (FBS) is being explored. These results will help to understand the origin of potential electrochemical current changes and the ideal experimental conditions to perform SECM measurements with living cells.[1] I. Beaulieu, S. Kuss, J. Mauzeroll, M. Geissler, Biological scanning electrochemical microscopy and its application to live cell studies, Anal. Chem. 83 (2011) 1485–1492. https://doi.org/10.1021/ac101906a.[2] D. Polcari, P. Dauphin-Ducharme, J. Mauzeroll, Scanning Electrochemical Microscopy: A Comprehensive Review of Experimental Parameters from 1989 to 2015, Chem. Rev. 116 (2016) 13234–13278. https://doi.org/10.1021/acs.chemrev.6b00067.[3] N. Thomas, D. Lima, D. Trinh, S. Kuss, Temperature Effect on the Electrochemical Current Response during Scanning Electrochemical Microscopy of Living Cells, Anal Chem. 95 (2023) 17962-17967. https://doi.org/10.1021/acs.analchem.3c03716

Dual excitation channel ratiometric fluorescent probes for visual and fluorescent detection of anthrax spore biomarker and tetracycline hydrochloride

Long-term and excessive use of tetracycline hydrochloride (TC) can lead to its accumulation in the environment, which can cause water contamination, bacterial resistance, and food safety problems. 2,6-Pyridine dicarboxylic acid (DPA) is a major biomarker of Bacillus anthracis spores, and its rapid and sensitive detection is of great significance for disease prevention and counter-terrorism. A bifunctional ratiometric fluorescent nanoprobe has been fabricated to detect DPA and TC. 3,5-dicarboxyphenylboronic acid (BOP) was intercalated into layered europium hydroxide (LEuH) by the ion-exchange method and exfoliated into nanosheets as a fluorescent nanoprobe (PNP). DPA and TC could significantly enhance the red fluorescence of Eu3+ through the antenna effect under different excitation wavelengths, while the fluorescence of BOP can be used as a reference based on the constant emission intensity, realizing ratiometric detection. A low limit of detection (LOD) for the target (DPA: 9.7 nM, TC: 21.9 nM) can be achieved. In addition, visual detection of DPA and TC was realized using color recognition software based on the obvious color changes. This is the first ratiometric fluorescent nanoprobe based on layered rare-earth hydroxide (LRH) for the detection of DPA and TC simultaneously, which opens new ideas in the design of multifunctional probes.

Core-Shell Interface Engineering Strategies for Modulating Energy Transfer in Rare Earth-Doped Nanoparticles.

Rare earth-doped nanoparticles (RENPs) are promising biomaterials with substantial potential in biomedical applications. Their multilayered core-shell structure design allows for more diverse uses, such as orthogonal excitation. However, the typical synthesis strategies-one-pot successive layer-by-layer (LBL) method and seed-assisted (SA) method-for creating multilayered RENPs show notable differences in spectral performance. To clarify this issue, a thorough comparative analysis of the elemental distribution and spectral characteristics of RENPs synthesized by these two strategies was conducted. The SA strategy, which avoids the partial mixing stage of shell and core precursors inherent in the LBL strategy, produces RENPs with a distinct interface in elemental distribution. This unique elemental distribution reduces unnecessary energy loss via energy transfer between heterogeneous elements in different shell layers. Consequently, the synthesis method choice can effectively modulate the spectral properties of RENPs. This discovery has been applied to the design of orthogonal RENP biomedical probes with appropriate dimensions, where the SA strategy introduces a refined inert interface to prevent unnecessary energy loss. Notably, this strategy has exhibited a 4.3-fold enhancement in NIR-II in vivo imaging and a 2.1-fold increase in reactive oxygen species (ROS)-related photodynamic therapy (PDT) orthogonal applications.

Activatable near-infrared fluorescent/photoacoustic probe for rapid identification of β-lactam-resistant bacteria

Early identification of antimicrobial-resistant (AMR) bacteria is of great significance to guide the use of drugs and reduce the risk of AMR infection. One of the main reasons for AMR resistance to antibiotic therapy is the production of β-lactamase that decomposes antibiotics. Accurate and rapid detection of β-lactamase is crucial o deal with bacterial resistance. Herein, a dual-modal probe with near-infrared fluorescence (NIRF) and photoacoustics (PA) to rapid detection of β-lactamases (TEM-1) and β-lactam-resistant bacteria (BLRB) was ingeniously designed and developed. The NIRF and PA signal of CyPA-L can be fully turned on after 7 min of incubation of CyPA-L with TEM-1, and also showed high selectivity for TEM-1 among multiple interfering analytes. Notably, a portable PA device with immediate detection (POCT) capability was developed to detect β-lactamase and BLRB through PA signaling of CyPA-L. The design of probe and facile device will provide a new way for the diagnosis and symptomatic treatment of early drug-resistant bacteria.

Design of deep-red emissive forced intercalation-induced light-up peptide as an indicator for the HIV-1 TAR RNA-ligand assay: integration of benzo[c,d]indole-quinoline (BIQ) cyanine dye into Tat peptide.

We report on a deep-red emissive fluorogenic peptide probe for human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA as an indicator for fluorescence indicator displacement (FID) assay. The probe design is based on the concept of the forced intercalation of thiazole orange (TO) dyes (FIT) on the peptide backbone, as recently proposed by our group, where the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. Here, instead of green emissive TO, we utilized a deep-red emissive benzo[c,d]indole-quinoline (BIQ) cyanine dye developed previously by our group for imaging of nucleolar RNA in living cells. The developed 9-mer FIT peptide (RKKRR-BIQ-RRR; named BIQ-FiLuP) exhibits a significant off-on signaling ability for TAR RNA (λem = 660nm, I/I0 = 130-fold, Φfree = 0.0009, Φbound = 0.052), and the dissociation constant Kd reaches ca. 1nM. When used in FID assay, BIQ-FiLuP, like TO-based FiLuP, is able to distinguish between competitive and noncompetitive inhibitors, which has never been demonstrated with all previous indicators for TAR RNA. Deep-red emissive BIQ-FiLuP facilitates the evaluation of green to yellow emissive ligands without suffering from optical interference. The combination use with green emissive TO-based FiLuP (λem = 541nm) would cover the examination of a wide range of fluorescent test compounds.

A DNA-peptide probe coupled mass spectrometry-based method for the detection of LncRNA in hepatocyte and serum samples

Liver cancer, a prominent contributor to cancer-related deaths, necessitates timely identification for successful intervention. In recent years, long non-coding RNAs (lncRNAs) have gained attention as potential biomarkers for liver cancer, and accurate quantification of lncRNA is essential for early diagnosis of liver cancer. However, existing methods for lncRNA detection rely on amplification and labeling techniques, resulting in semi-quantitative or qualitative outcomes. DNA-peptide technology offers a promising alternative. It has successfully translated nucleic acid quantification into peptide quantification, particularly in the context of miRNA analysis. However, the intricate structure of long-chain lncRNAs presents a challenge not encountered with miRNAs, making the design and acquisition of specific DNA-peptide probes for direct quantification more difficult. In this study, a strategy for lncRNA quantification was developed using a DNA-peptide probe coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quasi-targeted proteomics. The inherent structural complexity of long-chain lncRNAs was overcome by designing DNA-peptide probes based on their unique sequence segments, enabling direct quantification without the need for amplification. Highly upregulated in liver cancer (HULC) was identified as a target lncRNA through bioinformatics analysis using GEO2R online software. Subsequently, the target lncRNA (i.e., HULC), which had been biotinylated and bound to streptavidin agarose beforehand, was hybridized with the probe. Following trypsin digestion, the reporter peptide was released and quantified using LC-MS/MS. Finally, this method was applied to analyze HULC levels in liver cancer cell lines and serum samples, enabling quantitative assessment and early diagnosis. The results demonstrated that liver cancer diagnostics could be enhanced through the integration of mass spectrometry and DNA-peptide probe technology for lncRNA quantification.