Biosensors and Bioelectronics

Sensitive phenotyping of serum extracellular vesicles on a SERS-microfluidic platform for early-stage clinical diagnosis of ovarian carcinoma

Ovarian carcinoma (OvCa) poses a severe threat to women's health due to its high mortality rate and lack of efficient early diagnosis approach. There is evidence to suggest that nanosized small extracellular vesicles (sEVs) which carrying cell-specific components from OvCa can serve as potential diagnostic biomarkers. Herein, we reported a Surface-enhanced Raman Scattering (SERS)-multichannel microchip for sEVs (S-MMEV) assay to investigate the phenotype changes of sEVs. The microchip composed of seven microchannels, which enabled the parallel detection of multiple biomarkers to improve the detection accuracy. Using SERS probes conjugated with antibodies recognizing different biomarkers including ubiquitous EV biomarkers (i.e., tetraspanins; CD9, CD81) and putative OvCa tumor biomarkers (i.e. EpCAM, CD24, CA125, EGFR), we successfully analyzed the phenotypic changes of sEVs and accurately differentiated OvCa patients from healthy controls, even at early stage (I-II), with high sensitivity, high specificity and an area under the curve value of 0.9467. Additionally, the proposed approach exhibited higher sensitivity than conventional methods, demonstrating the efficiency of precise detection from cell culture and clinical samples. Collectively, the developed EV phenotyping approach S-MMEV could serve as a potential tool to achieve the early clinical diagnosis of OvCa for further precise diagnosis and personal treatment monitoring.

CRISPR/Cas14a integrated with DNA walker based on magnetic self-assembly for human papillomavirus type 16 oncoprotein E7 ultrasensitive detection

To enhance the biomarker diagnostics sensitivity and selectivity of human papillomavirus type 16 oncoprotein E7 (HPV16 E7) in serum, a label/enzyme-free electrochemical detection platform was developed. This platform featured a type of "Super-turn-off" nanobiosensor monitored through differential pulse voltammetry (DPV). It integrated the magnetic self-assembly property of the α-Fe

Design and application of prodrug fluorescent probes for the detection of ovarian cancer cells and release of anticancer drug

Ovarian cancer is a gynecologic malignancy with high mortality. The main reason is that it is detected at an advanced stage due to a lack of early diagnosis and treatment. Therefore, it is of great interest to develop a chemical tool that can visualize ovarian cancer cells in real-time and eliminate them. Unfortunately, probes that can simultaneously monitor both modes of action for the diagnosis and treatment of ovarian cancer have not been developed. Here, we designed a novel prodrug fluorescent probe (YW-OAc) that not only visually tracks cancer cells but also enables the on-demand delivery of chemotherapeutic agents. By β-Gal-mediated glycosidic bond hydrolysis, the fluorescent signal changed from blue to green (signal 1), enabling visual tracking of ovarian cancer cells. Subsequently, the identified cancer cells were subjected to precise light irradiation to induce anticancer drug release accompanied by a fluorescence transition from green to blue (signal 2), enabling real-time information on drug release. Thus, the prodrug fluorescent probe YW-OAc provides comprehensive two-step monitoring during cancer cell recognition and clearance. Notably, YW-OAc exhibited high affinity (K

Design and application of proximity hybridization-based multiple stimuli-responsive immunosensing platform for ovarian cancer biomarker detection

The development of convenient and sensitive multi-readout immunoassay is crucial but highly challenged for meeting the demand of exactness and diversity in early clinical diagnosis. Herein, a split-type multiple stimuli-responsive biosensor was outlined combined with the outstanding superiority of luminol probe-based electrochemiluminescence (ECL) strategy, mimicking enzyme-mediated colorimetric system and portable photothermal effect-induced temperature sensing. Especially, versatile MoS

Surface roughness-induced absorption acts as an ovarian cancer cells growth sensor-monitor

Uncontrolled growth of ovarian cancer cells is the fifth leading cause of female cancer deaths since most ovarian cancer patients are diagnosed at an advanced stage of metastatic disease. Here, we report on the sensor for monitoring the cancer treatment efficiency in real-time. We measure the optical interaction between the evanescent fields of microfiber and ovarian cancer inter-cellular medium at different treatment stages. Spectral absorption signatures are correlated with optical micrographs and western blot tests. We found that the treatment of tumor cells with induces both cells growth arrest and alter the spectral lines in a dose-dependent manner. These observations are mediated by surface roughness out of silica glass material, form an essential step toward the development of early detection of response to cancer therapy.

A novel electrochemical immunosensor for ultrasensitive detection of CA125 in ovarian cancer

In the current study, we report on the design and development of a novel electrochemical immunosensor for the detection of cancer antigen 125 (CA125) oncomarker. Polyamidoamine/gold nanoparticles (PAMAM/AuNPs) were used to increase the conductivity and enhance the number of antibodies (Abs) immobilized on the electrode surface. Three-dimensional reduced graphene oxide-multiwall carbon nanotubes (3DrGO-MWCNTs) were used to modify the glassy carbon electrode to improve the electrode conductivity and specific surface area. Ab and toluidine blue attached to O-succinyl-chitosan-magnetic nanoparticles (Suc-CS@MNPs) as a tracer. The poor solubility of chitosan (CS) was improved by succinic anhydride using a novel modification method. Under optimum condition, the developed immunosensor exhibited a wide linear range (0.0005-75 U/mL) and an excellent limit of detection around 6 μU/mL. The reliability of the engineered immunosensor in detecting CA125 was verified by standard addition recovery method, which was further compared to enzyme-linked immunosorbent assay (ELISA). The proposed immunosensor exhibited excellent stability, high selectivity and sensitivity, and good reproducibility. Based on the great performance of the engineered immunosensor, it is proposed as a robust and reliable diagnostic tool for the detection of CA125 in the clinic.

Dual-cycle signal amplification on an Au@CeO2-x/Bi2MoO6 platform: Toward an ultrasensitive and enzyme-free sandwich immunosensor for CA125 in clinical diagnostics

An electrochemical biosensor for sensitive detection of CKAP4 with application to the diagnosis of ovarian cancer

Cytoskeletal remodeling is crucial in tumor progression and metastasis, with cytoskeleton-associated protein 4 (CKAP4) being a key protein involved this process; however, its detection remains a challenge. In this paper, we report a solid-state electrochemistry-enhanced biosensor based on Cu-TCPP nanosheets for sensitive detection of CKAP4. In the meantime, the proposed biosensor can present high specificity in CKAP4 recognition. Specifically, upon recognition of the target, the designed catalytic hairpin assembly (CHA) reaction is successfully initiated, generating lots of DNA duplexes, which can be cleaved by Exo III to release numerous truncated thiolated signal DNA (sDNA). Subsequently, the generated sDNA is introduced to a high-conductivity electrode surface modified with Cu-TCPP nanosheets which are loaded with methylene blue. Moreover, the introduction of sDNA triggers ligand displacement via competitive coordination on this surface, thereby forming non-electroactive Cu-sDNA complexes, which significantly modulates the current signals, enabling sensitive quantification of CKAP4 levels. The biosensor may achieve a low detection limit of 0.30 pg/mL, with a broad linear range covering five orders of magnitude, and exhibits satisfying anti-interference ability in complex biological matrices. Furthermore, it displays outstanding discriminatory accuracy in differentiating ovarian cancer patient samples from healthy controls, showing great potential for cancer diagnosis.

Interface self-shelling effect-mediated photoinduced carrier transport and multiplexed signal amplification mechanism in self-powered photoelectrochemical biosensing

In the realm of biomedical diagnostics, the development of sensitive and specific detection methods for cancer biomarkers is of paramount importance. Herein, we report on the design and implementation of a self-powered photoelectrochemical (PEC) sensor that harnesses amplified photocathode signals for the deterioration of carbohydrate antigen 125 (CA125) associated with ovarian cancer. This self-powered sensing platform integrates Cu

Highly integrated and compact transduction strategy for multidimensional sensing: Near infrared light mediated transmission of optical, electrical and visual signals for ovarian cancer marker assay

Mediating effective signal conversion strategies through intermediate parameters to develop easily integrated smart sensing platforms remains a challenge. Herein, a compact and miniaturized optical, electrical and visual sensor was constructed through reasonably designing and assembling three sub-sensors, which can simultaneously and independently feedback signal changes brought by near-infrared (NIR) light. Concretely, the thermal effect induced by multifunctional V

Magnetic Fe3O4-Au@UIO-66-NH2@toehold probe mediated fluorescent sensor for detecting ovarian cancer-specific circRNA coupled with hybridization chain reaction and the CRISPR-Cas12a system

Cooperative amplification of Prussian blue as a signal indicator and functionalized metal-organic framework-based electrochemical biosensor for an ultrasensitive HE4 assay

Human epididymis protein 4 (HE4), a diagnostic biomarker of ovarian cancer, is crucial for monitoring the early stage of the disease. Hence, it is highly important to develop simple, inexpensive, and user-friendly biosensors for sensitive and quantitative HE4 assays. Herein, a new sandwich-type electrochemical immunosensor based on Prussian blue (PB) as a signal indicator and functionalized metal-organic framework nanocompositesas efficient signal amplifiers was fabricated for quantitative analysis of HE4. In principle, ketjen black (KB) and AuNPs modified on TiMOF (TiMOF-KB@AuNPs) could accelerate electron transfer on the electrode surface and act as a matrix for the immobilization of antibodies via cross-linking to improve the determination sensitivity. The PB that covalently binds to labeled antibodies endows the biosensors with intense electrochemical signals. Furthermore, the concentration of HE4 could be indirectly detected by monitoring the electroactivity of PB. Benefiting from the high signal amplification ability of the PB and MOF nanocomposites, this strategy displayed a wide linear range (0.1-80 ng mL

β-Galactosidase-activated near-infrared AIEgen for ovarian cancer imaging in vivo

Near-infrared (NIR) aggregation induced-emission luminogens (AIEgens) circumvent the noisome aggregation-caused quenching (ACQ) effect in physiological milieu, thus holding high promise for real-time and sensitive imaging of biomarkers in vivo. β-Galactosidase (β-Gal) is a biomarker for primary ovarian carcinoma, but current AIEgens for β-Gal sensing display emissions in the visible region and have not been applied in vivo. We herein propose an NIR AIEgen QM-TPA-Gal and applied it for imaging β-Gal activity in vitro and in ovarian tumor model. After being internalized by ovarian cancer cells (e.g., SKOV3), the hydrophilic nonfluorescent QM-TPA-Gal undergoes hydrolyzation by β-Gal to yield hydrophobic QM-TPA-OH, which subsequently aggregates into nanoparticles to turn NIR fluorescence "on" through the AIE mechanism. In vitro experimental results indicate that QM-TPA-Gal has a sensitive and selective response to β-Gal with a limit of detection (LOD) of 0.21 U/mL. Molecular docking simulation confirms that QM-TPA-Gal has a good binding ability with β-Gal to allow efficient hydrolysis. Furthermore, QM-TPA-Gal is successfully applied for β-Gal imaging in SKOV3 cell and SKOV3-bearing living mouse models. It is anticipated that QM-TPA-Gal could be applied for early diagnosis of ovarian cancers or other β-Gal-associated diseases in near future.

Cervical cancer screening by biomarker-free Serum-SERS technique: A three-principal-substrate approach

The complexity of serum constituents and the lack of knowledge on biomarkers bring obstacles for reliable cancer screening with the limited information obtained by surface-enhanced Raman scattering (SERS) on a single substrate. We report here an extremely high-accurate approach for cancer screening by biomarker-free serum-SERS technique which employs three principal SERS substrates that are of specially designed surface properties. With three substrates rich information on serum can be obtained from SERS spectra, of which quadratic discriminant analysis (QDA) yields excellent classifications of serum samples independently. Diagnosis with an accuracy of ∼100% can be achieved based on the three independent QDA classifications, by the unanimity principle. In this study, a screening accuracy of 100% was achieved by this approach for 122 diagnosed out of the 194 serum samples. The mechanism for the extremely high accuracy of this approach was also theoretically investigated. The three-principal-substrate approach opens up a path towards highly accurate biomarker-free cancer screening by serum-SERS technique.

An electrochemical biosensor designed with entropy-driven autocatalytic DNA circuits for sensitive detection of ovarian cancer-derived exosomes

Intelligent artificial DNA circuits have emerged as a promising approach for modulating signaling pathways and signal transduction through rational design, which may contribute to comprehensively realizing biomolecular sensing of organisms. In this work, we have fabricated an electrochemical biosensor for the sensitive and accurate detection of ovarian cancer-derived exosomes by constructing an entropy-driven autocatalytic DNA circuit (EADC). Specifically, the robust EADC is prepared by the self-assembly of well-designed DNA probes, and upon stimulation of the presence of ovarian cancer cells-derived exosomes, numerous inputs can be produced to feedback and accelerate the reaction. The catalytic abilities of the generated input sequences play a pivotal role in EADC and dramatically enhance the signal amplification capability. Through the combination of the autocatalytic circuit and circular cleavage reactions, significantly changed electrochemical signals can be recorded for sensitive analysis of the exosomes with a remarkably low detection limit of 30 particles/μL. Moreover, the proposed enzyme-free biosensor shows exceptional performance in distinguishing patient samples from healthy samples, which exhibits promising prospects for the clinical diagnosis of ovarian cancer.

Ultrasensitive detection of miRNA with single-nucleotide resolution using a high-electron-mobility transistor biosensor combined with splint-ligation

The use of microRNAs as clinical cancer biomarkers is hindered by the absence of accurate, sensitive and rapid assays for their detection in biofluids. Here we report a biosensing approach, SpLig-HEMT, that combines an RNA splint-ligation reaction with an AlGaN/GaN high-electron-mobility transistor biosensor for ultrasensitive miRNA detection. In this system, HEMT functions as a highly effective voltage amplifier to enhance detection sensitivity, while the splint-ligation reaction ensures precise discrimination of single-nucleotide mutations. By detecting miRNA-21, the SpLig-HEMT biosensor achieves an exceptional limit of detection of 10

Integration of label-free surface enhanced Raman spectroscopy (SERS) of extracellular vesicles (EVs) with Raman tagged labels to enhance ovarian cancer diagnostics

We report a proof-of-concept diagnostic strategy that integrates multiplexed Raman-tagged antibody labeling with label-free surface-enhanced Raman spectroscopy (SERS) and machine learning (ML) to improve the detection of ovarian cancer via extracellular vesicles (EVs). EVs were isolated from patient plasma using size-exclusion chromatography and labeled with polyyne-based Raman tags targeting three ovarian cancer biomarkers: CA-125, HE4, and CA-19-9. Labeled and unlabeled EVs were deposited onto SERS-active substrates, and spectra were collected using a custom confocal Raman microscope. Incorporating the tag-derived signal into SERS analysis enhanced interpretability and added molecular specificity. We evaluated classification performance using various ML models applied to spectral datasets from a cohort of ovarian cancer patients and healthy controls. Combined use of the Raman tag and label-free regions improved classification accuracy compared to either modality alone. Notably, support vector machine (SVM) achieved over 95 % accuracy, sensitivity, and specificity. Compared to ELISA, our SERS platform demonstrated improved sensitivity in detecting EV-associated biomarkers from small sample volumes. This approach addresses a key limitation of SERS-based diagnostics by linking spectral features to known biomarkers, offering improved transparency and performance in ML-enabled liquid biopsy.

Nano biosensor unlocks tumor derived immune signals for the early detection of ovarian cancer

Ovarian cancer is a critical health issue for women nowadays. Its impact is significant because of its high mortality rate (324,603 worldwide), late-stage diagnosis and poor survival rate. Lack of screening tests, vague symptoms, misdiagnosis, and age factor makes it even more difficult to detect. Neutrophils, a subset of immune cells, undergo tumor-specific changes as ovarian cancer progresses inside ovarian tumour microenvironment. Therefore, monitoring the time-specific activity of neutrophils in circulation has the potential to aid in the diagnosis of ovarian cancer. Most ovarian tumor-specific antigens are unknown, making it difficult to identify neutrophils associated with ovarian tumor. We present ovarian tumor-associated circulating neutrophil cell profiling as a stand-alone cancer diagnostic method using a liquid biopsy. Using a SERS-functionalized nano probe, the metabolic profiles of neutrophils from ovarian tumor interaction are detected. We demonstrate that neutrophils associated with cancer stem cells have a distinct metabolic profile and are useful in the diagnosis of early ovarian cancer. Using 5 μL of peripheral blood and an artificial neural network, the characteristics of neutrophil profiles in patient blood could distinguish cancer cohort from non-cancer (healthy) with a 90 % sensitivity and 100 % specificity. Our results demonstrate the viability of using circulating neutrophils for non-invasive cancer diagnostics.

CRISPR-Cas12a-integrated pregnancy test strip biosensors: Visual detection of telomerase and miRNA let-7a in cervical cancer diagnostics

Cervical cancer is a leading cause of female cancer-related mortality globally, and early screening based on reliable biomarkers is critical for improving prognosis. Telomerase (a key driver of cellular immortalization) and microRNA let-7a (a tumor suppressor with downregulated expression in cervical cancer) are well-validated diagnostic targets, but existing detection methods are hindered by complex procedures, high instrumentation costs, and reliance on specialized technical expertise-limiting their accessibility in resource-constrained settings. To address these limitations, we developed two novel CRISPR-Cas12a-integrated biosensors using commercially available pregnancy test strips (PTS) for instrument-free, visual readout. Both biosensors leverage a core signal mediator, probe 1 ("MB-ssDNA1-hCG"), which links CRISPR-Cas12a activation to visible color development on the PTS. The first Biosensor CRISPR-PTS-Telo detects telomerase activity in one-step without PCR: telomerase-generated (TTAGGG)n repeats activate Cas12a-crRNA1 complex, cleaving the probe 1 to release hCG, achieving a detection limit of 18 HeLa cells-comparable to sensitive laboratory assays. The second Biosensor CRISPR-PTS-let7a detects miRNA let-7a by first converting miRNA signals to Trigger DNA via Assister DNA and probe 2 ("MB-ssDNA2+Trigger"), activating Cas12a-crRNA2 complex, cleaving the probe 1 and inducing PTS coloration. This achieves a detection limit of 25.1 fM for let-7a. Validation with clinical samples (24 cervical tissues and 26 blood samples) confirmed their concordance with gold-standard methods (ELISA for telomerase, RT-qPCR for let-7a). These versatile tools hold significant potential as point-of-care testing (POCT) solutions to facilitate early, accessible cervical cancer screening.

T7-assisted special rolling circular amplification platform for point-of-care cervical cancer screening

Cervical cancer, primarily caused by high-risk human papillomavirus (HPV) infections, remains a leading cause of mortality among women in underdeveloped and developing countries. Conventional screening methods, necessitating professional equipment and trained personnel, are impractical in resource-limited settings, underscoring the need for a point-of-care (POC) detection platform. Isothermal nucleic acid amplification techniques (NAATs) are widely used in POC applications due to their cost-effectiveness and instrument-free nature. In this study, a novel one-pot dual amplification system, T7-MSSRCA was developed, by integrating the minimum secondary structure rolling circular amplification (MSS-RCA) with T7 Exonuclease. This system achieved an amplification time of 15 min and a total diagnostic time of approximately 1 h. Moreover, signal detection using both fluorescence and lateral flow assay (LFA) strips was achieved through reporter probe modifications. By employing a human chorionic gonadotropin (hCG)-modified reporter probe, the T7-MSSRCA system facilitated quantitative POC detection via pregnancy test strips. The system demonstrated impressive sensitivities for synthesized HPV16 targets, achieving 1 fM with fluorescent output and 10 fM with paper strips. When tested with 50 cervical swab samples, T7-MSSRCA exhibited a sensitivity of 0.95 and a specificity of 0.9333 compared to RT-PCR measurements.

Rapid and ultrasensitive electrochemical detection of DNA methylation for ovarian cancer diagnosis

Alterations in DNA methylation, a stable epigenetic marker, are important components in the development of cancer. It is vital to develop diagnostic systems with the ability to rapidly quantify DNA methylation with high sensitivity and selectivity. However, the analysis of DNA methylation must address two main challenges: (i) ultralow abundance and (ii) differentiating methylated cytosine from normal cytosine on target DNA sequence in the presence of an overwhelming background of circulating cell-free DNA. Here we report the development of an ultrasensitive and highly-selective electrochemical biosensor for the rapid detection of DNA methylation in blood. The sensing of DNA methylation involves the hybridization on a network of probe DNA modified gold-coated magnetic nanoparticles (DNA-Au@MNPs) complementary to target DNA, and subsequently enzymatic cleavage to differentiate methylated DNA strands from corresponding unmethylated DNA strands. The biosensor presents a dynamic range from 2 aM to 20 nM for 110 nucleotide DNA sequences containing a single-site methylation with the lowest detected concentration of 2 aM. This DNA-Au@MNPs based sensor provides a promising method to achieve 35 min response time and minimally invasive diagnosis of ovarian cancer.