Unveiling Colorectal Cancer Biomarkers: Harnessing Biosensor Technology for Volatile Organic Compound Detection.

Conventional screening options for colorectal cancer (CRC) detection are mainly direct visualization and invasive methods including colonoscopy and flexible sigmoidoscopy, which must be performed in a clinical setting and may be linked to adverse effects for some patients. Non-invasive CRC diagnostic tests such as computed tomography colonography and stool tests are either too costly or less reliable than invasive ones. On the other hand, volatile organic compounds (VOCs) are potentially ideal non-invasive biomarkers for CRC detection and monitoring. The present review is a comprehensive presentation of the current state-of-the-art VOC-based CRC diagnostics, with a specific focus on recent advancements in biosensor design and application. Among them, breath-based chromatography pattern analysis and sampling techniques are overviewed, along with nanoparticle-based optical and electrochemical biosensor approaches. Limitations of the currently available technologies are also discussed with an outlook for improvement in combination with big data analytics and advanced instrumentation, as well as expanding the scope and specificity of CRC-related volatile biomarkers.

An In Vitro Study of Saffron Carotenoids: The Effect of Crocin Extracts and Dimethylcrocetin on Cancer Cell Lines.

Crocus sativus L. has various pharmacological properties, known for over 3600 years. These properties are attributed mainly to biologically active substances, which belong to the terpenoid group and include crocins, picrocrocin and safranal. The aim of the current work was to examine the effects of crocins (CRCs) and their methyl ester derivate dimethylcrocetin (DMCRT) on glioblastoma and rhabdomyosarcoma cell lines, in terms of cytotoxicity and gene expression, implicated in proapoptotic and cell survival pathways. Cell cytotoxicity was assessed with Alamar Blue fluorescence assay after treatment with saffron carotenoids for 24, 48 and 72 h and concentrations ranging from 22.85 to 0.18 mg/mL for CRCs and 11.43 to 0.09 mg/mL for DMCRT. In addition, BAX, BID, BCL2, MYCN, SOD1, and GSTM1 gene expression was studied by qRT-PCR analysis. Both compounds demonstrated cytotoxic effects against glioblastoma and rhabdomyosarcoma cell lines, in a dose- and time-dependent manner. They induced apoptosis, via BAX and BID upregulation, MYCN and BCL-2, SOD1, GSTM1 downregulation. The current research denotes the possible anticancer properties of saffron carotenoids, which are considered safe phytochemicals, already tested in clinical trials for their health promoting properties.

Evaluation of Cancer Cell Lines by Four-Point Probe Technique, by Impedance Measurements in Various Frequencies.

Cell-based biosensors appear to be an attractive tool for the rapid, simple, and cheap monitoring of chemotherapy effects at a very early stage. In this study, electrochemical measurements using a four-point probe method were evaluated for suspensions of four cancer cell lines of different tissue origins: SK-N-SH, HeLa, MCF-7 and MDA-MB-231, all for two different population densities: 50 K and 100 K cells/500 µL. The anticancer agent doxorubicin was applied for each cell type in order to investigate whether the proposed technique was able to determine specific differences in cell responses before and after drug treatment. The proposed methodology can offer valuable insight into the frequency-dependent bioelectrical responses of various cellular systems using a low frequency range and without necessitating lengthy cell culture treatment. The further development of this biosensor assembly with the integration of specially designed cell/electronic interfaces can lead to novel diagnostic biosensors and therapeutic bioelectronics.

Clinical Application of the Novel Cell-Based Biosensor for the Ultra-Rapid Detection of the SARS-CoV-2 S1 Spike Protein Antigen: A Practical Approach.

The availability of antigen tests for SARS-CoV-2 represents a major step for the mass surveillance of the incidence of infection, especially regarding COVID-19 asymptomatic and/or early-stage patients. Recently, we reported the development of a Bioelectric Recognition Assay-based biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus in just three minutes, with high sensitivity and selectivity. The working principle was established by measuring the change of the electric potential of membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody after attachment of the respective viral protein. In the present study, we applied the novel biosensor to patient-derived nasopharyngeal samples in a clinical set-up, with absolutely no sample pretreatment. More importantly, membrane-engineered cells were pre-immobilized in a proprietary biomatrix, thus enabling their long-term preservation prior to use as well as significantly increasing their ease-of-handle as test consumables. The plug-and-apply novel biosensor was able to detect the virus in positive samples with a 92.8% success rate compared to RT-PCR. No false negative results were recorded. These findings demonstrate the potential applicability of the biosensor for the early, routine mass screening of SARS-CoV-2 on a scale not yet realized.

Development of a Portable, Ultra-Rapid and Ultra-Sensitive Cell-Based Biosensor for the Direct Detection of the SARS-CoV-2 S1 Spike Protein Antigen.

One of the key challenges of the recent COVID-19 pandemic is the ability to accurately estimate the number of infected individuals, particularly asymptomatic and/or early-stage patients. We herewith report the proof-of-concept development of a biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus. The biosensor is based on membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody. We demonstrate that the attachment of the protein to the membrane-bound antibodies resulted in a selective and considerable change in the cellular bioelectric properties measured by means of a Bioelectric Recognition Assay. The novel biosensor provided results in an ultra-rapid manner (3 min), with a detection limit of 1 fg/mL and a semi-linear range of response between 10 fg and 1 µg/mL. In addition, no cross-reactivity was observed against the SARS-CoV-2 nucleocapsid protein. Furthermore, the biosensor was configured as a ready-to-use platform, including a portable read-out device operated via smartphone/tablet. In this way, we demonstrate that the novel biosensor can be potentially applied for the mass screening of SARS-CoV-2 surface antigens without prior sample processing, therefore offering a possible solution for the timely monitoring and eventual control of the global coronavirus pandemic.

Bioelectrical Analysis of Various Cancer Cell Types Immobilized in 3D Matrix and Cultured in 3D-Printed Well.

Cancer cell lines are important tools for anticancer drug research and assessment. Impedance measurements can provide valuable information about cell viability in real time. This work presents the proof-of-concept development of a bioelectrical, impedance-based analysis technique applied to four adherent mammalian cancer cells lines immobilized in a three-dimensional (3D) calcium alginate hydrogel matrix, thus mimicking in vivo tissue conditions. Cells were treated with cytostatic agent5-fluoruracil (5-FU). The cell lines used in this study were SK-N-SH, HEK293, HeLa, and MCF-7. For each cell culture, three cell population densities were chosen (50,000, 100,000, and 200,000 cells/100 µL). The aim of this study was the extraction of mean impedance values at various frequencies for the assessment of the different behavior of various cancer cells when 5-FU was applied. For comparison purposes, impedance measurements were implemented on untreated immobilized cell lines. The results demonstrated not only the dependence of each cell line impedance value on the frequency, but also the relation of the impedance level to the cell population density for every individual cell line. By establishing a cell line-specific bioelectrical behavior, it is possible to obtain a unique fingerprint for each cancer cell line reaction to a selected anticancer agent.

An Ultra-Rapid Biosensory Point-of-Care (POC) Assay for Prostate-Specific Antigen (PSA) Detection in Human Serum.

Prostate-specific antigen (PSA) is the established routine screening tool for the detection of early-stage prostate cancer. Given the laboratory-centric nature of the process, the development of a portable, ultra rapid high-throughput system for PSA screening is highly desirable. In this study, an advancedpoint-of-care system for PSA detection in human serum was developed based on a cellular biosensor where the cell membrane was modified by electroinserting a specific antibody against PSA. Thirty nine human serum samples were used for validation of this biosensory system for PSA detection. Samples were analyzed in parallel with a standard immunoradiometric assay (IRMA) and an established electrochemical immunoassay was used for comparison purposes. They were classified in three different PSA concentration ranges (0, <4 and ≥4 ng/mL). Cells membrane-engineered with 0.25 µg/mL anti-PSA antibody demonstrated a statistically lower response against the upper (≥4 ng/mL) PSA concentration range. In addition, the cell-based biosensor performed better than the immunosensor in terms of sensitivity and resolution against positive samples containing <4 ng/mL PSA. In spite of its preliminary, proof-of-concept stage of development, the cell-based biosensor could be used as aninitiative for the development of a fast, low-cost, and high-throughput POC screening system for PSA.

Development and validation of a cellular biosensor detecting pesticide residues in tomatoes.

Two of the most important categories of pesticides used in agricultural practice are organophosphates and dithiocarbamates. Their extensive and inappropriate use has rendered their reliable monitoring at trace levels more and more necessary. This study presents the construction of a rapid and sensitive cellular biosensor test based on the measurement of changes of the cell membrane potential of immobilized cells, according to the working principle of the Bioelectric Recognition Assay (BERA). The cells were immobilized by entrapment in a sodium alginate bead and directly applied in different pesticide dilutions and agricultural samples. The pesticides used were the organophosphate insecticide diazinon and the dithiocarbamate fungicide propineb. Two different cell types, N2a (neuroblastoma) and Vero (fibroblast) were used as the biosensory elements in order to investigate their differential response against the pesticides. In this way, we hoped to increase the selectivity of the assay. Based on the observed patterns of response, we demonstrate that the sensor can be used for the qualitative and, in some concentrations, quantitative detection of the pesticides with a high degree of reproducibility. The lowest detected concentration was 3nM. Finally, for the investigation of the effects of different pesticides on the accumulation of cytosolic Ca(2+), we conducted a fluorescent assay on N2a cells treated with tomato sample extracts, which were replicates of the E.U. proficiency test sample. The tomato samples were either organically grown or contained 14 different pesticides. The experimental results showed a higher increase of the intracellular Ca(2+) concentration in cells treated with non-organic samples compared to the cells treated with organic samples.

Assessment of Organophosphate and Carbamate Pesticide Residues in Cigarette Tobacco with a Novel Cell Biosensor.

The conventional analysis of pesticide residues in analytical commodities, such as tobacco and tobacco products is a labor intensive procedure, since it is necessary to cover a wide range of different chemicals, using a single procedure. Standard analysis methods include extensive sample pretreatment (with solvent extraction and partitioning phases) and determination by GC and HPLC to achieve the necessary selectivity and sensitivity for the different classes of compounds under detection. As a consequence, current methods of analysis provide a limited sample capacity. In the present study, we report on the development of a novel cell biosensor for detecting organophosphate and carbamate pesticide residues in tobacco. The sensor is based on neuroblastoma N2a cells and the measurement of changes of the cell membrane potential, according to the working principle of the Bioelectric Recognition Assay (BERA). The presence of pesticide residues is detected by the degree of inhibition of acetylcholine esterase (AChE). The sensor instantly responded to both the organophoshate pesticide chlorpyriphos and the carbamate carbaryl in a concentration-dependent pattern, being able to detect one part per billion (1 ppb). Additionally, tobacco leaf samples (in blended dry form) were analyzed with both the novel biosensor and conventional methods, according to a double-blind protocol. Pesticide residues in tobacco samples caused a considerable cell membrane hyperpolarization to neuroblastoma cells immobilized in the sensor, as indicated by the increase of the negative sensor potential, which was clearly distinguishable from the sensor's response against pesticide-free control samples. The observed response was quite reproducible, with an average variation of +5,6%. Fluorescence microscopy observations showed that treatment of the cells with either chlorpyrifos or carbaryl was associated with increased [Ca²+]cyt . The novel biosensor offers fresh perspectives for ultra-rapid, sensitive and low-cost monitoring of pesticide residues in tobacco as well as other food and agricultural commodities.