Analytica Chimica Acta

Salt-induced gold nanoparticles aggregation lights up fluorescence of DNA-silver nanoclusters to monitor dual cancer markers carcinoembryonic antigen and carbohydrate antigen 125

In clinical diagnosis of cancer, the monitoring of single tumor marker may result in many false and missed results, while simultaneous detection of multiple tumor markers should be more accuracy and effective. Here, we report a new strategy that salt-induced gold nanoparticles (AuNPs) aggregation lights up fluorescence of dual-color DNA-silver nanoclusters-aptamer (DNA-AgNCs-apta) for the simultaneous monitoring of carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA125). The dual-color aptasensor system is composed of green-emitting DNA-AgNCs with CEA aptamer (gDNA

Synergistic effect of the combination therapy on ovarian cancer cells under microfluidic conditions

Ovarian cancer belongs to the group of gynecological cancers and indicates the high resistance to many drugs used in standard anticancer therapy. The treatment of ovarian cancer is a big challenge for the present medicine. In our report we tested the effectiveness of the combination anticancer therapy against ovarian cells: human ovarian carcinoma (A2780) and human ovarian fibroblasts (HOF). Two different types of drugs were used: doxorubicin (DOX) and a new-generation photosensitizer, nanoencapsulated meso-tetraphenylporphyrin (nano-TPP). The aim of the research was to compare the effect of the sequential combination therapy (chemotherapy with DOX and photodynamic therapy with nano-TPP) carried out in static and dynamic conditions. To achieve dynamic culture conditions, similar to in vivo environment, we designed a new microfluidic system in which the simultaneous, independent cultures of two cell lines (non-malignant and cancer cells) and their one-step analysis were possible. We observed that the sequential combination of photodynamic therapy (PDT) with chemotherapy allowed to obtain the synergistic effect of the treatment with using low doses of drugs. We also confirmed that the use of microfluidic conditions significantly increased the effectiveness of combination therapy and allowed for maintaining a high selectivity of the action of drugs on cancer cells. To the best of our knowledge, for the first time the microfluidic system was used to carry out sequential combination therapy against ovarian cancer.

Graphene oxide-based qRT-PCR assay enables the sensitive and specific detection of miRNAs for the screening of ovarian cancer

Circulating microRNAs (miRNAs) have the potential to become reliable and noninvasive biomarkers for ovarian cancer (OC) diagnosis; however, the conventional miRNAs detection techniques exhibit enduring limitations of low sensitivity and specificity. Graphene oxide (GO), a novel nanomaterial, is at the forefront of material design for extensive biomedical applications. Owing to the excellent water affinity and single-stranded DNA (ssDNA) adsorption characteristics of GO, we designed and developed a GO-based qRT-PCR assay for the detection of miRNAs associated with OC. In the GO-based qRT-PCR system, GO could significantly improve the sensitivity and specificity of the qRT-PCR assay by noncovalently interacting with primers and ssDNA and reducing the occurrence of non-specific amplification. Moreover, the detection of miRNAs associated with OC confirmed that GO-based qRT-PCR assay could differentiate benign ovarian tumors from OC (sensitivity, 0.91; specificity, 1.00). Collectively, these findings provide robust evidence that GO-based qRT-PCR assay can be effectively used as a promising method to detect miRNAs for the screening of OC patients.

Ultrasmall iron oxide nanoparticles cisplatin (IV) prodrug nanoconjugate: ICP-MS based strategies to evaluate the formation and drug delivery capabilities in single cells

Ultrasmall iron oxide nanoparticles (<10 nm) were explored here as nanotransporters of cis-diamminetetrachloroplatinum (IV) (a cisplatin prodrug) in cellular models. The coating of the particles containing reactive carboxylic acid groups enabled the formation of a stable conjugate between the prodrug and the nanoparticles using one pot reaction. The nanoconjugate was characterized by different techniques exhibiting diameters of about 6.6 ± 1.0 nm. The use of a hyphenated strategy based on high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) permitted the quantitative evaluation of Fe and Pt in the nanoconjugate. Furthermore, the cellular uptake of the synthetic nanoconjugate was explored by single cell-ICP-MS (SC-ICP) which was used for the first time in this type of studies. The experiments in A2780 and A2780cis, sensitive and resistant ovarian cancer cell models respectively, revealed intracellular platinum concentrations of 12 fg/cell and 4 fg/cell, respectively which were 4-fold higher with respect to the uptake of cisplatin in both models. Intracellular drug release from the nanoconjugate was proved by measuring DNA platination in the same cells. In this case, levels of about 250 ng Pt/mg DNA were observed, about 5-fold higher when the nanoconjugate was used in comparison to cisplatin. Furthermore, the differences between the two lines turned to be significantly smaller than in the case of using cisplatin. The quantitative analytical tools developed here provided essential information required to fully characterize the developed nanoplatforms particularly important to overcome drug resistance.

Photoacoustic/fluorescence dual-modality cyanine-based probe for real-time imaging of endogenous cysteine and in situ diagnosis of cervical cancer in vivo

Cysteine (Cys), one of cellular biothiols in the organism, is associated with many diseases, such as Parkinson's disease, Alzheimer's disease, liver damage, rheumatoid arthritis, and cancer. However, activatable fluorescence/photoacoustic (FL/PA) probes for non-invasive, real-time, and deep imaging of Cys in vivo are still lacking, and this hinders the diagnosis of Cys-related diseases. Herein, we designed and synthesized a novel activated FL/PA dual-modality cyanine-base probe (FP

Highly water-soluble AIEgen for α-amylase activity ultrasensitive detection and ovarian cancer rapid theranostic

Salivary α-amylase, as a digestive enzyme, is associated with variations in levels related to stress, diet, and certain medical conditions. Research indicates that it may have potential applications in assessing physiological and psychological stress responses. However, the clinical application of salivary amylase is still in the exploratory stage, lacking large-scale standardized testing methods. On the other hand, early theranostic of ovarian cancer remains a significant challenge, as traditional ultrasound examinations and tumor markers have limited effectiveness and insufficient specificity. Overall, both fields continue to evolve under clinical application and research drive, but further validation and improvement are still needed. In this study, we designed and synthesized a highly water-soluble AIEgen TPAG with a galactose structure, which not only could be used for ultrasensitive detection of α-amylase activity in saliva, but also for rapid diagnosis and therapy of ovarian cancer. Specifically, the limit of detection of α-amylase activity detection curve constructed based on TPAG absorbance changes was 0.004749 U/mL, and the limit of quantitation was 0.01439 U/mL. Even compared with commercial kits, the detection error still did not exceed 5 %, which further demonstrates the high reliability of this method. This method provides an effective tool for evaluating human sensory experience through the fluctuation of salivary α-amylase activity. Meanwhile, due to TPAG could be rapidly identified and taken up significantly by overexpressed β-galactosidase receptor in ovarian cancer, which providing a new tool for rapid theranostic of ovarian cancer. Salivary α-amylase detection and ovarian cancer screening are two important areas in current medical research. This innovative technology not only provides a fast and convenient detection method for monitoring salivary amylase levels, but also helps evaluate individuals' physiological and psychological stress states to promote the development of high-quality tobacco. It can also improve the early detection and precision therapy of ovarian cancer and make up for the limitations of traditional detection methods.

Development of a high-throughput kinase activity platform using nanoLC-MS/MS with DIA approach for studying the anti-cancer mechanism of Taxol in ovarian cancer

Protein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward. Herein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. This platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy.

Fluorescence turn-on immunosensing of HE4 biomarker and ovarian cancer cells based on target-triggered metal-enhanced fluorescence of carbon dots

Rapid and sensitive detection of tumor biomarkers and cancer cells is of crucial importance for the early diagnosis and prognosis prediction of cancer. The present report describes a target-induced fluorescence enhancement immunosensor that utilizes the optical property of carbon dots (CDs) and the metal-enhanced fluorescence effect (MEF) property of silver nanoparticles (AgNPs) for the sensitive detection of the cancer biomarker human epididymis protein 4 (HE4) and ovarian cancer cells. Nitrogen and sulfur co-doped CDs with a quantum yield of 85.6% were prepared and served as the fluorophore in MEF. The HE4 antibody (Ab) specific to the HE4 antigen was linked covalently to the surface of the synthesized CDs as the capture. The HE4 Ab-conjugated AgNPs (AgNPs-Ab) were prepared and utilized as signal amplification elements. In the presence of the target HE4, composite sandwich structures were formed between the labeled CDs-Ab and AgNPs-Ab, which brought the CDs and AgNPs into proximity, resulting in the fluorescence of CDs enhancement owing to MEF. The intensity of fluorescence enhancement was positively correlated with the HE4 concentration in the clinically important range of 0.01-200 nM with a limit detection of 2.3 pM. Moreover, the immunosensor was also successfully applied to specific fluorescence labeling and quantitative determination of HE4-positive ovarian cancer cells. The proposed target-triggered MEF sensor platform demonstrated high sensitivity, excellent anti-interference ability, along with successful validation in complex biological matrices, providing a new approach for HE4 detection in early diagnosis and therapeutic monitoring.

Engineering a G-quadruplex-based logic gate platform for sensitive assay of dual biomarkers of ovarian cancer

As most single tumor markers have low sensitivity or specificity, the joint detection of multiple tumor biomarkers is helpful to improve the positive rate and specificity of early diagnosis. To establish a sensitive, specific, and rapid screening method for ovarian cancer, two enzyme-free logic gates were developed to realize the joint detection of carbohydrate antigen 125 (CA125) and carcinoembryonic antigen (CEA). G-quadruplex with peroxidase activity was used as a chromogenic catalyst in this colorimetric diagnosis. For NOR gate, in the absence of CEA and CA125, template DNA (Tem-DNA), CEA aptamer with half of the G-quadruplex sequence (CEA-apt), and CA125 aptamer with the other half of the G-quadruplex sequence (CA125-apt) form a double-stranded DNA equipped with one G-quadruplex structure which can strongly combine with hemin to obtain the peroxidase-like activity; here, the output of this NOR gate is set as 1, which could exclude the risk of ovarian cancer. For AND gate, CEA-apt and CA125-apt are separated from magnetic beads in the co-existence of the dual biomarkers. After adding Tem-DNA, the G-quadruplex structure constructs with hemin to exhibit the output as 1, presenting a high risk of ovarian cancer. Under the optimized condition, this novel assay can not only show a sharp color difference between physiological state and pathological state, but also be employed for the quantitative analysis of every single biomarker. The linear detection range and detection limit for CEA are 5-500 ng mL

Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters

Cancer is known as one of the main non-communicable diseases and the leading cause of death in the new era. Early diagnosis of cancer requires the identification of special biomarkers. Currently, microRNAs (miRNAs) have attracted the attention of researchers as useful biomarkers for cancer early detection. Hence, various methods have been recently developed for detecting and monitoring miRNAs. Among all miRNAs, detection of miRNA-21 (miR-21) is important because it is abnormally overexpressed in most cancers. Here, a new biosensor based on silver nanoclusters (AgNCs) is introduced for detecting miR-21. As a fluorescent probe, a rationally designed hairpin sequence containing a poly-cytosine motif was used to facilitate the formation of AgNCs. A guanine-rich sequence was also employed to enhance the sensing signal. It was found that in the absence of miR-21, adding a guanine-rich sequence to the detecting probe caused only a slight change in the fluorescence emission intensity of AgNCs. While in the presence of miR-21, the emission signal enhanced. A direct correlation was observed between the increase in the fluorescence of AgNCs and the concentration of miR-21. The performance of the proposed biosensor was characterized thoroughly and confirmed. The biosensor detected miR-21 in an applicable linear range from 9 pM to 1.55 nM (LOD: 2 pM). The designed biosensor was successfully applied for detecting miR-21 in human plasma samples and also in human normal and lung and ovarian cancer cells. This biosensing strategy can be used as a model for detecting other miRNAs. The designed nanobiosensor can measure miR-21 without using any enzymes, with fewer experimental steps, and at a low cost compared to the reported biosensors in this field.

An aptamer integrated electrochemistry and photoluminescence dual-mode microfluidic biosensor for sensitive and accurate ovarian cancer extracellular vesicles detection

Extracellular vesicles (EVs) serve as crucial biomarkers for cancer screening due to their close association with the physiological and pathological states of cancer cells. These membrane-bound vesicles carry molecular cargo that reflects the characteristics of their parent cells, making them valuable diagnostic indicators. Current EVs detection methods face significant limitations in clinical applications, particularly regarding sensitivity and accuracy requirements for reliable cancer diagnostics. The critical challenge is to develop sensitive and accurate EVs detection methods for clinical cancer screening. We developed an aptamer-integrated dual-mode microfluidic biosensor combining electrochemistry (EC) and photoluminescence (PL) detection for ovarian cancer EVs screening. The platform utilized aptamer-functionalized gold nanoflowers (Au NFs) integrated with 3D laser-induced graphene (LIG) electrode arrays, achieving a 1.44-fold increase in electroactive surface area. Poly-lysine (PLL) served as the PL detection substrate for capturing fluorescent complexes, enabling simultaneous dual-signal generation. Under optimized conditions, the biosensor achieved a detection range of 10-10 This work presents the first aptamer-integrated EC-PL dual-mode microfluidic biosensor for EVs detection, enabling real-time signal cross-validation and enhanced reliability. The novel combination of Au NFs-3D LIG architecture with PLL-mediated detection strategy significantly improves sensitivity and clinical applicability for point-of-care cancer screening.

Hybrid chain reaction and selective recognition-based homogeneous dual-fluorescence analysis of circulating tumor cells in clinical ovarian cancer samples

Oncological analysis is important in tumor diagnosis. We constructed a dual-fluorescence and binary visual analysis system for circulating tumor cells (CTCs) using the folate receptor as a biomarker, combined with hybridization chain reaction and nanomaterial amplification. This strategy integrates terminal protection, selective recognition properties of N-methyl mesoporphyrin IX and CdTe quantum dots for Cu In fluorescence mode, folate receptor and A2780 ovarian cancer cells were specifically detected with a limit of detection of 0.1 fg mL This dual-fluorescence and binary visual CTCs detection method provides multiple options for clinical tumor liquid biopsy.

Rational design of genotyping nanodevice for HPV subtype distinction

There are more than 200 subtypes of human papillomavirus (HPV), and high-risk HPVs are a leading cause of cervical cancer. Identifying the genotypes of HPV is significant for clinical diagnosis and cancer control. Herein, we used programmable and modified DNA as the backbone to construct fluorescent genotyping nanodevice for HPV subtype distinction. In our strategy, the dye-labeled single-stranded recognize-DNA (R-DNA) was hybridized with Black Hole Quencher (BHQ) labeled single-stranded link-DNA (L-DNA) to form three functionalized DNA (RL-DNA). Through the extension of polycytosine (poly-C) in L-DNA, three RL-DNAs can be more firmly adsorbed on graphene oxide to construct reliable genotyping nanodevice. The genotyping nanodevice had low background noise since the dual energy transfer, including Förster resonance energy transfer (FRET) from dye to BHQ and the resonance energy transfer (RET) from dye to graphene oxide. Meanwhile, the programmability of DNA allows the proposed strategy to simultaneously and selectively distinguish several HPV subtypes in solution using DNA labeled with different dyes. To demonstrate clinical potential, we show multiplexed assay of HPV subtypes in cervical scrapes, and it has been successfully applied in HPV-DNA analysis in cervical scrapes samples. The genotyping nanodevice could be developed for simultaneous and multiplex analysis of several oligonucleotides in a homogeneous solution by adjusting the recognition sequence, demonstrating its potential application in the rapid screening of multiple biomarkers.

An amplification-free method for the detection of HOTAIR long non-coding RNA

The discovery of large transcripts of long RNAs that have limited protein coding capacity, known as long non-coding RNAs (lncRNAs) present new concepts on RNA-mediated gene regulation. Increasing evidence suggests that large intervening ncRNAs regulate key pathways in cancer genesis and metastasis. Among the most characterized lncRNAs, homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) acts as an oncogenic molecule in different cancer cells, and thus its expression level serves as a potential biomarker for diagnostic and therapeutic purposes in several human cancers, such as breast, prostate, liver and ovarian cancer. This paper reports a simple and sensitive sensor platform for the detection of HOTAIR. Extracted HOTAIR sequences from ovarian cancer cells and plasma samples derived from ovarian cancer patients were magnetically isolated and purified, followed by a sandwich hybridization event at a screen-printed gold electrode. This event was monitored by amperometry using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H

Coupling metal-organic framework nanosphere and nanobody for boosted photoelectrochemical immunoassay of Human Epididymis Protein 4

A "signal-on" photoelectrochemical (PEC) immunosensor for highly sensitive detection of Human Epididymis Protein 4 (HE4), a new serum biomarker of ovarian cancer with small molecular weight, was fabricated by coupling the porphyrin-based metal-organic framework (MOF) nanosphere (nPCN-224) and Nanobody (Nb). To label the Nb, the nPCN-224 with an average size of 160-200 nm was prepared by solvothermal method. The mechanism for the photocurrent generation of nPCN-224 was systematically investigated, showing that the dissolved O

Label-free electrochemical immunosensor for picomolar detection of the cervical cancer biomarker MCM5

An immunosensor for label-free electrochemical detection of MiniChromosome Maintenance Protein 5, MCM5, a protein overexpressed in cervical cancer, based on a gold electrode is reported. The electrode was first modified with a submonolayer (capture layer) of 11-mercaptoundecanoic acid (11-MUA) and then activated with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to immobilize the capture antibody. The change in electrode surface properties (wettability) during the formation of the 11-MUA layers was determined using the static water contact angle (WCA). The binding of MCM5 antigens to the capture antibody was monitored by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using 5 mM [Fe(CN)