Isothermal titration calorimetry (ITC) is a powerful label-free method for quantifying protein-DNA interactions, providing direct measurements of binding affinity, stoichiometry, enthalpy, and entropy in a single experiment. However, the quality of protein-DNA ITC data depends strongly on experimental details, including accurate concentration determination, careful buffer matching, sample quality, instrument cleanliness, sample loading, degassing, and calibration. Without careful control, these factors can obscure true binding behavior and complicate thermodynamic interpretation. Here, we present a practical checklist for designing, optimizing, and troubleshooting ITC experiments for protein-DNA binding studies. Using homeodomain transcription factor-DNA interactions as representative examples, we identify common failure modes, including concentration errors, buffer mismatch, poorly optimized binding-partner concentrations, sample carryover, cell or syringe contamination, loading artifacts, and calibration problems. For each potential issue, we outline how to recognize the problem, choose appropriate controls, and apply corrective strategies to improve data quality and reproducibility. Together, this checklist provides a practical framework for improving protein-DNA ITC experiments by helping researchers distinguish genuine binding behavior from correctable technical artifacts and generate more reliable thermodynamic measurements.
Methylglyoxal (MGO) is a reactive endogenous α-dicarbonyl involved in glycation, oxidative stress, and metabolic disease. Its measurement in biological samples remains challenging because conventional assays generally require derivatization, chromatographic separation, and costly instrumentation. Fluorescence detection offers a simpler alternative, but currently available probes often lack selectivity for MGO over other biologically relevant carbonyl compounds. We therefore developed a series of N-substituted 4-hydrazino-7-nitrobenzofurazan derivatives for fluorimetric detection of MGO. Alkyl groups were introduced on the hydrazine nitrogen to modulate probe reactivity, steric accessibility, and the fluorescent properties of the resulting hydrazones. The probes were characterized by fluorescence spectroscopy, HPLC with fluorescence detection, LC-MS, and density functional theory calculations. Compared with unsubstituted NBD-H, most N-substituted derivatives produced stronger and more stable responses to MGO, while showing weaker responses to competing carbonyl compounds. Chromatographic and mass spectrometric analyses confirmed formation of the expected probe-MGO hydrazones, and calculations supported preferential condensation at the aldehyde group of MGO. In human plasma, most N-substituted probes retained a strong and highly linear fluorescence response to MGO, even in the presence of biologically relevant carbonyl interferents such as glyoxal, acrolein, acetaldehyde, and formaldehyde. Compared with unsubstituted NBD-H, these derivatives markedly improved analytical sensitivity toward MGO, supporting their use for fluorimetric detection in complex biological matrices. These findings support a microplate-compatible fluorimetric assay for MGO requiring only a protein-precipitation step and avoiding chromatographic separation or extensive sample clean-up.
Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) are two major immunosuppressive oncogenic viruses that severely threaten the global poultry industry. Field co-infection of these two pathogens is prevalent, accompanied by highly similar pathological lesions, making conventional diagnostic technologies incapable of rapid on-site differential identification. Recombinase polymerase amplification (RPA) coupled with the CRISPR/Cas12a system represents an innovative platform for rapid nucleic acid detection. In this study, target-specific crRNAs were designed against the conserved unique genes of MDV and REV, respectively, to establish a CRISPR-based diagnostic system capable of differentiating the two viruses for the first time. Comprehensive optimization of RPA reaction composition, buffer formulation and incubation temperature improved the overall detection performance. Three independent visual signal readout modalities were supported by this assay, including real-time fluorescence quantification, naked-eye fluorescence visualization under blue light, and lateral flow assay (LFA). Sensitivity evaluation demonstrated that the limit of detection (LOD) reached 1×100 copies/μL for MDV and 1×101 copies/μL for REV. Specificity assays verified that the system only generated specific positive signals in response to target viruses without cross-reactivity against other common avian oncogenic viruses and their subgroups. Blind testing of 30 clinical chick fecal specimens revealed that the assay achieved a 100% positive detection rate for both viruses when referenced to qPCR and RT-qPCR, with outstanding diagnostic efficacy and perfect detection consistency. Collectively, the developed RPA-CRISPR/Cas12a assay overcomes the limitations of traditional diagnostic methods and holds great promise for on-site routine screening in poultry farms and epidemiological surveillance of MDV and REV.
The simultaneous determination of short-chain fatty acids (SCFAs) and amino acids is of increasing interest, as these metabolite classes are important indicators of host metabolism and gut microbiota activity. They also serve as biomarkers for disease diagnosis and indicators of food composition. In this study, a gas chromatography-mass spectrometry method was developed for the simultaneous determination of both substituted (hydroxy-, amino- and hydroxy-amino-) and unsubstituted SCFAs, alongside amino acids, following derivatization and liquid-liquid extraction. Isobutyl chloroformate was employed as the derivatization reagent. The proposed method demonstrated excellent linearity, with coefficients of determination (R2) ranging from 0.9840 to 0.9992. Method limits of detection ranged from 0.011 to 7.2 μg/mL, while method limits of quantification ranged from 0.033 to 8.0 μg/mL. Intra-day precision (%RSD) ranged from 1.0% to 6.5% for biological samples and from 1.1% to 4.5% for eggs. Inter-day precision (%RSD) ranged from 1.0% to 5.6% for biological samples and from 2.0% to 6.7% for eggs. Matrix effects ranged from 98% to 108% for biological samples and from 96% to 113% for eggs, while recoveries ranged from 90% to 111% and from 95% to 108%, respectively. To the best of our knowledge, this is the first GC-MS method enabling the simultaneous determination of substituted and unsubstituted SCFAs together with amino acids in a single analytical procedure. The proposed approach provides a reliable and versatile platform for metabolomics, microbiome research, clinical investigations, and food analysis, while also providing considerable potential for expansion to a wider range of target metabolites.
The gold standard for SARS-CoV-2 neutralization assays involves wild-type virus, which requires Biosafety Level 3 (BSL-3) containment. To improve safety and accessibility, pseudovirus-based neutralization assays utilizing non-replicating particles like Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike protein can be conducted under BSL-2 conditions. This study aimed to perform the analytical validation of a VSV-based pseudovirus system for SARS-CoV-2 using recombinant monoclonal antibody. Pseudo-VSV carrying SARS-CoV-2 Spike proteins were produced using LentiX-293T cells. The assay system was optimized for Multiplicity of Infection (MOI) and assessed for specificity, limit of quantification (LOQ), linearity, accuracy, and precision using the neutralizing mAb BD-604. The system was optimized at an MOI of 0.25. The assay proved highly specific, as mAb BD-604 showed clear neutralizing activity while mAb 1A9 did not. The limit of quantification (LOQ) was determined to be 125 ng of mAb BD-604, with a linear range of 125 - 1000 ng. The relative accuracy remained within the 80-120% range, and the precision (%CV) ranged from 1.92% to 13.57%. Additionally, the system successfully characterized variant-specific neutralization, revealing that BD-604 was effective against the Wuhan, Delta, and Omicron BA.1/BA.2 strains but lacked activity against the Omicron XBB.1.5 variant. This validated pseudo-VSV based SARS-CoV-2 neutralization assay is a valuable bioassay for evaluating neutralizing antibody potency against various SARS-CoV-2 strains in a BSL-2 environment, thus making it useful for vaccine and therapeutic development.
The biological potency of recombinant human growth hormone (rhGH) is a critical quality attribute (CQA) for ensuring clinical efficacy. However, current pharmacopoeial methods, such as the USP in vitro Nb2-11 cell proliferation assay, lack specificity as they measure lactogenic activity via prolactin receptors rather than the growth hormone receptor (GHR). In this study, we developed a highly specific and robust potency assay for rhGH by standardizing and validating a murine pro-B lymphocyte (Ba/F3) cell platform stably expressing human GHR (hGHR). While GHR-transfected cell models have been previously described for basic research, this work bridges the gap between research-grade tools and pharmaceutical quality control (QC). We established a strictly characterized monoclonal cell line to ensure the long-term genetic and functional stability essential for routine commercial testing. The engineered Ba/F3-hGHR cells exhibited a dose-dependent proliferative response exclusively triggered by hGHR signaling. Through systematic optimization of assay parameters-including cell seeding density, serum concentrations, and incubation kinetics-we established a high-performance analytical protocol. The method was rigorously validated according to ICH Q2(R2) guidelines, demonstrating superior intermediate precision (GCV 8.4-18.3%), high accuracy, and the sensitivity required to detect subtle potency shifts in degraded samples. Our findings provide a mechanism-based, validated alternative to current regulatory methods, offering a reliable approach for the accurate quantification of rhGH biological activity in biopharmaceutical manufacturing.
Isolation of extracellular vesicles (EVs) from milk is technically challenging due to the presence of high concentrations of casein and other proteinaceous contaminants. In this study, we report an optimized two-step polyethylene glycol (PEG) precipitation protocol designed to improve the recovery and enrichment of camel milk-derived EVs. The performance of this two-step protocol, in which initial precipitation at PEG10% is followed by re-precipitation at Re-PEG5%, was evaluated and compared against several alternative isolation strategies, including single-and two-step PEG10% precipitation, and PEG precipitation combined with ultrafiltration (UF) and size-exclusion chromatography (SEC), with protocols assessed across multiple parameters. A suite of quantitative assessments, including protein-to-lipid and nucleic acid-to-protein ratio measurements and flow cytometry, was performed alongside qualitative characterizations via TEM, SEM, AFM, ATR-FTIR, NMR, DSC, EDS and elemental mapping. Complementary characterizations consistently demonstrated that the two-step Re-PEG5% protocol generated EV preparations with higher purity and reduced co-isolation of non-vesicular contaminants than the two-step PEG10% protocol or protocols involving costlier UF and SEC techniques. This improved purity was accompanied by enhanced cytocompatibility in HEK293 cells, as verified through MTT assays, suggesting that optimization of PEG concentration is a critical determinant of both camel milk EV quality and biological performance. Overall, the physicochemical and biological characterizations performed provide substantial evidence supporting the quality and yield of the EVs isolated from camel milk using a two-step PEG precipitation protocol. This cost-effective and scalable strategy offers a promising, green-chemistry-aligned alternative for EV production in biomedical applications, particularly in resource-limited settings.
Effective brucellosis control necessitates rapid serological screening assays suitable for on-site deployment. B. neotomae, a smooth Brucella species handled under lower-containment conditions, is a potential alternative source of lipopolysaccharide (LPS) antigen. This study aimed to develop and evaluate a competitive colloidal gold immunochromatographic strip based on B. neotomae LPS for detecting antibodies against B. melitensis and B. abortus in cattle and goats. A screened LPS-reactive monoclonal antibody, mAb 1B4, was incorporated into a dual-label format with an independent control line. The strip detected Brucella Positive National Standard Antiserum at 5.0 IU/mL, whereas the detection limit of the Rose Bengal test (RBT) exceeded 20 IU/mL. No cross-reactivity was observed with the limited panel of sera positive for Escherichia coli O157, Salmonella Dublin, Yersinia enterocolitica O:9, or rough Brucella. The strip also showed good repeatability and stability. Using commercial competitive ELISA (cELISA) as the confirmatory method, overall agreement was 97.37% ((kappa = 0.94) for goat sera and 98.75% ((kappa = 0.98) for cattle sera, both higher than the corresponding values for RBT. These findings indicate that the B. neotomae LPS-based strip has the potential to serve as a rapid preliminary screening assay for detecting antibodies against B. melitensis and B. abortus in cattle and goats. Further validation using larger, geographically diverse serum panels with well-defined infection and vaccination status is warranted before broader field application.
Reverse transcription quantitative PCR (RT-qPCR) is widely used in wastewater-based epidemiology (WBE) but is frequently compromised by matrix-associated inhibition. While mitigation strategies abound, a systematic framework for diagnosing RT-qPCR inhibition in wastewater remains lacking. Here, a mechanistic classification framework for RT-qPCR inhibitors in wastewater is established based on two orthogonal dimensions: kinetic effect (linear vs. exponential inhibition) and molecular target (nucleic acid sequestrators vs. enzyme activity inhibitors). This framework enables diagnosis of the dominant inhibitor types in a given sample and predicts the efficacy of mitigation strategies. Guided by this framework, the following findings are demonstrated: (1) sample dilution effectively relieves enzyme activity inhibition but fails to address nucleic acid sequestrators; (2) two-step RT-qPCR—by decoupling reverse transcription and PCR amplification—effectively circumvents RT-qPCR inhibition under the tested conditions. Notably, supplementation with T4 gene 32 protein (gp32), a single-stranded DNA-binding protein predicted by the framework to selectively relieve RNA sequestrators, did not produce consistent improvement across wastewater samples—a result that, within the diagnostic logic of the framework, implicates enzyme inhibitors as the predominant inhibitory species in our sample set. The deinhibition rate, introduced here as a quantitative metric, varied predictably with wastewater characteristics, extraction method, and target RNA concentration, with high-abundance RNA viruses showing disproportionately stronger effects. These findings provide a theoretical foundation and practical guidance for improving the accuracy and reliability of RT-qPCR-based wastewater surveillance.
The Organisation for the Prohibition of Chemical Weapons (OPCW) organizes the annual Trial Biotoxins Proficiency Test (ToxPT) to implement standardized and transparent evaluation on the technical proficiency of laboratories applying for or retaining official designation for biotoxin analysis. Laboratories are required to pass all relevant ToxPT events to prove sustained detection capability. Launched in August 2025, the Second Trial ToxPT centered on ricin detection and marked the first inclusion of high-molecular-weight protein toxins within an OPCW proficiency test. This study aimed to construct an immunological detection platform for ricin using conventional analytical methods, including WB and ELISA, and to evaluate their application performance in the Second Trial ToxPT. By optimizing the experimental parameters of immunoassays-including antibody coating concentration, blocking buffer selection, and working concentration of enzyme-labeled secondary antibody-we successfully established WB and sandwich ELISA methods for ricindetection. As a quantitative method, the ELISA assay employed four-parameter nonlinear regression to fit the standard curve and derive the standard equation, with a correlation coefficient (R2) of 0.9974, a limit of detection (LOD) of 1 ng/mL, a limit of quantitation (LOQ) of 7 ng/mL, and a broad quantitative range of 7-400 ng/mL. When applied to sample analysis in the second trial ToxPT, the obtained results were accurate and consistent with official reference data. These validated methods exhibit high reliability for routine ricin detection and lay a solid technical foundation for laboratories to sustain their biotoxin analysis accreditation.
Protoporphyrin IX (PPIX) is a photoreactive heme precursor and is a key pathogenic driver in erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP). In both EPP and XLP, excess PPIX in plasma circulates to the skin and hepatobiliary system, resulting in acute, painful cutaneous photosensitivity and in some patients, gallstones, or hepatic failure. A key distinguishing feature between EPP and XLP is proportions of PPIX that become metalated with zinc (Zn-PPIX) or remain metal-free. Thus, ability to accurately measure both PPIX and Zn-PPIX in plasma is especially useful for assessing emerging therapies for EPP and XLP. This manuscript presents a fully validated, sensitive liquid chromatography-tandem mass spectrometry method for the quantification of PPIX and Zn-PPIX across a dynamic range in human plasma, which is especially suited for measuring drug-induced effects on plasma PPIX and Zn-PPIX levels. It also presents the first reported accurate measurements of plasma PPIX in healthy individuals and begins to establish a preliminary baseline range for this major form of PPIX in plasma of individuals without protoporphyria, which can facilitate future development of therapies for EPP and XLP.
The interaction of doxorubicin (DOX) - anthracycline antibiotic routinely used in the cancer treatment - with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) has been studied by electroanalysis. As a part of the study, the redox behavior of DOX on carbon screen printed electrodes was thoroughly investigated by cyclic and square wave voltammetry. Preparations of DNA differing in molecular size and secondary structure, PCR-generated dsDNA fragments, and synthetic oligonucleotides and their duplex were employed as examples of ssDNA and dsDNA. Prior to studying, DNA samples were carefully characterized to verify DNA structure. The binding strength of DOX to both ssDNA and dsDNA under physiological pH and close to physiological ionic strength was found to be rather weak (with the binding constants of the order of 103-104 L/mol) and undistinguishable in practical terms. That suggests the electrostatic attraction between positively charged molecules of DOX and the negatively charged phosphate groups of DNA chains as a major driving force for the DOX-DNA interactions. Other modes of interaction such as the specific binding into minor and major grooves of dsDNA and the DOX insertion between paired nucleobases may be secondary in relation to electrostatic interactions of DOX with DNA phosphate groups. The use of ssDNA as a special control in a study of drug-DNA interaction mechanisms appears as an essential requirement to correctly evaluate the contributions of various modes of binding to the overall binding strength.
Given the hepatotoxicity and widespread contamination of Aflatoxin B1 (AFB1), developing ultra-sensitive, anti-interference analytical platforms is paramount for public health. Herein, a dual signal ratio electrochemical sensing platform for accurate AFB1 analysis was constructed integrating DNA tetrahedrons (TNDA), a dynamic DNA walker, and nitrogen-doped graphene oxide-supported hollow silver-platinum bimetallic nanospheres (NGR-HP-AgPt). The NGR-HP-AgPt cavity provides a uniform microenvironment, synergistically promoting catalytic effects to significantly accelerate interfacial electron transfer. Simultaneously, rigid 3D TNDA prevent spatial probe entanglement, providing a well-oriented track. To avoid false-positive artifacts in complex matrices, a competitive binding strategy is employed. Upon target recognition, AFB1 displaces a complementary sequence (DNA1) from the aptamer. The released DNA1 acts as a walking strand, hybridizing with signal probes (Cd2+-DNA2). Subsequently, Exonuclease III initiates the DNA walker, continuously cleaving probes to amplify the ratiometric signal variation for reliable self-calibration. Under optimal conditions, this sensor exhibits a broad linear range from 2 × 10-4 to 20 ng/mL, with an ultra-low detection limit of 73.99 fg/mL. Furthermore, its practical utility and high accuracy were successfully validated in complex food and medicinal matrices, yielding consistent results with the standard HPLC-FL method. This work broadens the robust design paradigm of anti-interference biosensing.
Rapid and cost-effective detection of dengue infection, especially during the early stages of disease and in resource-limited settings, remains a significant public health concern. Recent advances in nanomaterial-based biosensors have provided promising opportunities for the development of simple, sensitive, and affordable nucleic acid detection platforms. In the present study, a Cu2+-modified reduced graphene oxide (Cu2+/rGO) nanostructure-based colorimetric biosensor integrated with hybridization chain reaction (HCR) was developed and assessed for the detection of a dengue virus target sequence. The nanozyme exhibited peroxidase-like activity and catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), generating a visible colorimetric signal. Following HCR-mediated double-stranded DNA formation, the catalytic activity of the nanostructure was suppressed, resulting in reduced color intensity and enabling signal-off detection. The proposed biosensor showed a linear response over 0.5-10 nM, with detection limits of 1.36 nM in buffer and 1.6 nM in human serum samples spiked with synthetic target DNA. The color variation was directly visible to the naked eye. The assay showed good linearity (R2 = 0.9868) while avoiding the need for thermocyclers and complex instrumentation. The Cu2+/rGO nanozyme preserved nearly 79% of its catalytic performance after 10 days of storage. Overall, the results suggest that the developed platform may provide a practical and cost-effective biosensing strategy for preliminary nucleic acid screening in resource-limited settings and complement conventional polymerase chain reaction (PCR)-based assays.
Dried suckling deer is an animal-derived traditional Chinese medicinal material that is vulnerable to adulteration in the marketplace because of its limited availability, relatively high price, and lack of unified quality standards. Therefore, reliable and rapid authentication of dried suckling deer is essential. This research aimed to establish rapid PCR-based colloidal gold immunochromatographic assay (PCR-GICA) and PCR-based fluorescent quantum dot immunochromatographic assay (PCR-FQICA) methods based on molecular authentication technology for the identification of dried suckling deer. The mtDNA ND4L gene of dried suckling deer was selected as the target gene, and dried suckling deer-specific primers were designed to amplify a 46 bp fragment. PCR amplification was performed, and the amplified products were rapidly detected using PCR-GICA and PCR-FQICA, with agarose gel electrophoresis used for verification. The specificity, reproducibility, and sensitivity of the two methods were evaluated. The results showed that the short-fragment primers designed from the mtDNA ND4L gene achieved the best specificity at an annealing temperature of 56 °C. Both PCR-GICA and PCR-FQICA showed positive results for the dried suckling deer reference material and authentic dried suckling deer samples, whereas the confusing product samples and the blank controls showed negative results. Both methods showed good specificity and reproducibility, and the lowest detectable concentration of template DNA was 0.5 pg/μL, which was 10-fold lower than that of agarose gel electrophoresis. In conclusion, the rapid identification methods established in this study are specific, sensitive, rapid, low-cost, and visual, and are suitable for the rapid identification of dried suckling deer.
This study aimed to establish the molecular biology analytical methods based on the ladder-shaped melting temperature isothermal amplification (LMTIA) and the proofreading enzyme-mediated probe cleavage (Proofman), and two dual-LMTIA methods were developed for detection of H. diffusa and H. corymbosa as well as H. diffusa and H. pinifolia, the specific LMTIA primers and probes targeting the Internal Transcribed Spacer (ITS) sequences of H. diffusa, H. corymbosa, and H. pinifolia were designed. The Proofman-LMTIA technique was validated for specificity, optimized for temperature, determined for sensitivity and repeatability, and tested with commercial samples. The results showed that the established duplex Proofman-LMTIA method for H. diffusa vs. H. corymbosa could be finished in 30 min at the optimal temperature of 58°C with high specificity, the sensitivity of 10 pg/μL genomic DNAs H. corymbosa and the detection limit of 1%. For the other duplex system targeting H. diffusa vs. H. pinifolia, the amplification was finished within 20 min at the optimal temperature of 69°C with high specificity, the sensitivity was 50 pg/μL gDNA of H. pinifolia, and the detection limit was 1%. For the applicability test of the established duplex Proofman-LMTIA method, 10 commercial dried whole herb samples labeled as H. diffusa were analyzed. Among them, 3 samples tested positive for H. diffusa, 6 contained both H. diffusa and H. corymbosa, 1 contained H. corymbosa only, and H. pinifolia was not detected. Summarily, H. diffusa, H. corymbosa and H. pinifolia could be rapidly and accurately detected by the established Proofman-LMTIA methods.
The multiplex detection of nucleic acid biomarkers, including circulating tumor DNAs (ctDNAs) and microRNAs (miRNAs), holds promise for improving the accuracy of molecular diagnostic techniques. In this study, we developed a CRISPR/Cas9-based sensing platform capable of simultaneously detecting multiple nucleic acid biomarkers. This approach leverages the activation of the CRISPR/Cas9 system through the generation of monomeric substrates mediated by rolling circle amplification (RCA). Specifically, upon the presence of a specific target, the RCA process is initiated, producing concatemeric DNA products. By incorporating an endonuclease and complementary reporter molecules, the RCA products are cleaved into monomeric substrates. Subsequently, Cas9/sgRNA complexes recognize and bind these monomeric substrates, activating the CRISPR/Cas9 systems, which further cleave the substrates to produce amplified fluorescence signals. Compared to conventional RCA methods, which often suffer from product aggregation, the monomeric substrate generation strategy demonstrated enhanced sensing capabilities and analytical performance, with signal intensities nearly double those observed in traditional approaches. To enable multiplex detection, three distinct sets of padlock probes, reporter molecules, and sgRNAs were designed to selectively target three different nucleic acid sequences. The platform exhibited high sensitivity and simultaneous detection of multiple ctDNAs and miRNAs, achieving limits of detection (LODs) ranging from 1.28 fM to 1.50 fM for ctDNAs and 0.61 fM to 0.97 fM for miRNAs. Additionally, analyses of clinical serum samples confirmed the method's accuracy and applicability within complex biological matrices. Collectively, this work introduces a multiplex nucleic acid biomarker detection platform with significant potential for advancing molecular sensing and clinical diagnostic applications.
Circulating tumor cells (CTCs) are important biomarkers in liquid biopsy for early cancer screening, prognosis evaluation, treatment monitoring, and prediction of metastatic risk; however, their extremely low abundance and pronounced heterogeneity remain major barriers to sensitive and reliable analysis. Aptamers have emerged as promising recognition ligands for CTC targeting owing to their high affinity and specificity, facile synthesis, and straightforward chemical modification. Meanwhile, nanomaterials offer unique optical, electrical, magnetic, and thermal properties that facilitate signal amplification, target enrichment, and the construction of multifunctional platforms. The integration of aptamers and nanomaterials has thus provided a versatile framework for the development of high-performance CTC detection systems and targeted cancer theranostic platforms. This review summarizes recent progress in aptamer-functionalized nanomaterials for CTC-related applications, including aptamer selection and optimization, CTC capture, separation, detection, and imaging, as well as their expanded applications in photothermal therapy, photodynamic therapy, and drug delivery. Representative noble-metal, carbon-based, semiconductor, organic, and composite nanoplatforms are further compared, with an emphasis on their functional advantages and current limitations. Finally, outstanding challenges in analytical robustness, biosafety, in vivo delivery, and clinical translation are discussed, and future directions toward multiplexed analysis, dynamic monitoring, and personalized precision oncology are highlighted.
A novel, sensitive, and environmentally sustainable spectrofluorimetric method was developed and validated for eszopiclone quantification in pharmaceutical formulations and human plasma. The method exploits fluorescence quenching of erythrosin B upon ground-state complex formation with eszopiclone. Stern-Volmer analysis at three temperatures (298, 303, and 313 K) confirmed a static quenching mechanism, with thermodynamic parameters (ΔH = -35.20 kJ mol-1, ΔS = -4.03 J mol-1 K-1, ΔG = -34.00 to -33.94 kJ mol-1) revealing spontaneous, exothermic, enthalpy-driven binding. Job's plot established 1:1 M stoichiometry. Box-Behnken response surface methodology optimized critical parameters, yielding optimal conditions of pH 6.8, buffer volume 1.3 mL, erythrosin B concentration 12 μg/mL, and reaction time 4 min. The validated method demonstrated excellent linearity (0.1-4.0 μg/mL, r2 = 0.9997), outstanding sensitivity (LOD = 0.031 μg/mL; LOQ = 0.094 μg/mL), superior precision (intra-day RSD = 1.130%; inter-day RSD = 1.861%), and satisfactory accuracy (99.33 ± 1.12%). Application to pharmaceutical tablets (99.21 ± 0.306%) and spiked plasma samples (98.50-100.40%) confirmed broad analytical utility, with statistical equivalence to a reference HPLC method established by t- and F-tests. Green chemistry assessment using AGREE (0.74), MoGAPI (78%), BAGI (70), and RGB12 (87.2% whiteness) confirmed high environmental sustainability.
