Analytical validation and clinical utilization of K-4CARE™: a comprehensive genomic profiling assay with personalized MRD detection.

Background: Biomarker testing has gradually become standard of care in precision oncology to help physicians select optimal treatment for patients. Compared to single-gene or small gene panel testing, comprehensive genomic profiling (CGP) has emerged as a more time- and tissue-efficient method. This study demonstrated in-depth analytical validation of K-4CARE, a CGP assay that integrates circulating tumor DNA (ctDNA) tracking for residual cancer surveillance. Methods: The assay utilized a panel of 473 cancer-relevant genes with a total length of 1.7 Mb. Reference standards were used to evaluate limit of detection (LOD), concordance, sensitivity, specificity and precision of the assay to detect single nucleotide variants (SNVs), small insertion/deletions (Indels), gene amplification and fusion, microsatellite instability (MSI) and tumor mutational burden (TMB). The assay was then benchmarked against orthogonal methods using 155 clinical samples from 10 cancer types. In selected cancers, top tumor-derived somatic mutations, as ranked by our proprietary algorithm, were used to detect ctDNA in the plasma. Results: For detection of somatic SNVs and Indels, gene fusion and amplification, the assay had sensitivity of >99%, 94% and >99% respectively, and specificity of >99%. Detection of germline variants also achieved sensitivity and specificity of >99%. For TMB measurement, the correlation coefficient between whole-exome sequencing and our targeted panel was 97%. MSI analysis when benchmarked against polymerase chain reaction method showed sensitivity of 94% and specificity of >99%. The concordance between our assay and the TruSight Oncology 500 assay for detection of somatic variants, TMB and MSI measurement was 100%, 89%, and 98% respectively. When CGP-informed mutations were used to personalize ctDNA tracking, the detection rate of ctDNA in liquid biopsy was 79%, and clinical utility in cancer surveillance was demonstrated in 2 case studies. Conclusion: K-4CARE™ assay provides comprehensive and reliable genomic information that fulfills all guideline-based biomarker testing for both targeted therapy and immunotherapy. Integration of ctDNA tracking helps clinicians to further monitor treatment response and ultimately provide well-rounded care to cancer patients.

A paper-based colorimetric sensor array for discrimination and simultaneous determination of organophosphate and carbamate pesticides in tap water, apple juice, and rice.

A colorimetric paper-based sensor is proposed for the rapid monitoring of six major organophosphate and carbamate pesticides. The assay was constructed by dropping gold and silver nanoparticles on the hydrophilic zones of a paper substrate. The nanoparticles were modified by L-arginine, quercetin, and polyglutamic acid. The mechanism of sensing is based on the interaction between the pesticide and the nanoparticles. The color of nanoparticles changed during the interactions. A digital camera recorded these changes. The assay provided a unique response for each studied pesticide. This method can determine six individual pesticides including carbaryl, paraoxon, parathion, malathion, diazinon, and chlorpyrifos. The limit of detection for these pesticides were 29.0, 22.0, 32.0, 17.0, 45.0, and 36.0 ng mL-1, respectively. The assay was applied to simultaneously determine the six studied pesticides in a mixture using the partial least square method (PLS). The root mean square errors of prediction were 11, 8.7, 9.2, 10, 12, and 11 for carbaryl, paraoxon, parathion, malathion, diazinon, and chlorpyrifos, respectively. The paper-based device can differentiate two types of studied pesticide (organophosphate and carbamate) as well as two types of organophosphate structures (oxon and thion). Furthermore, this sensor showed high selectivity to the pesticides in the presence of other potential species (e.g., metal ions, anions, amino acids, sugar, and vitamins). This assay is capable of determining the pesticide compounds in tap water, apple juice, and rice samples.Graphical abstract.

Well-Orientation Strategy for Direct Immobilization of Antibodies: Development of the Immunosensor Using the Boronic Acid-Modified Magnetic Graphene Nanoribbons for Ultrasensitive Detection of Lymphoma Cancer Cells.

This work presents an effective strategy for the well-oriented immobilization of antibodies in which boronic acid is directly attached to the surface and with no need of the long and flexible spacer. A magnetic graphene nanoribbon-boronic-acid-based immunosensor was developed and tested for the impedimetric detection of lymphoma cancer cells, a blood cancer biomarker. Magnetic graphene nanoribbons (MGNRs) were modified with boronic acid (BA) to create a supporting matrix that is utilized by immobilizing anti-CD20 antibodies with good orientation. The prepared biosensing layer (MGNR/BA/Ab) with well-oriented antibodies was premixed into whole blood samples to interact with lymphoma cancer cell receptors. In the presence of target cell receptors, an immunocomplex was formed between anti-CD20 antibodies and lymphoma cancer cell receptors. Then, the biosensing layer was magnetically collected on a screen-printed carbon electrode (SPCE) and placed in a homemade electrochemical cell configuration to measure impedimetric signals. The fabrication steps of the immunosensor were characterized by various techniques, such as resonance light scattering, fluorescence, electrochemical impedance spectroscopy, and cyclic voltammetry. The assay is highly sensitive: the calculated limit of detection of lymphoma cancer cells was as low as 38 cells/mL, and the detection was linear from 100 to 1 000 000 cells/mL. The specificity of the immunosensor is also very high, and there is no interference effect with several potential interferents, such as the breast cancer (MCF-7), human embryonic kidney (HEK293), and leukemia (HL-60 and KCL-22) cell lines. The performance of the immunosensor for lymphoma cancer cells in clinical blood samples is consistent with that of commercial flow cytometric assays.

Microfluidic chip with integrated electrical cell-impedance sensing for monitoring single cancer cell migration in three-dimensional matrixes.

Cell migration has been recognized as one hallmark of malignant tumor progression. By integrating the method of electrical cell-substrate impedance sensing (ECIS) with the Boyden chamber design, the state-of-the-art techniques provide kinetic information about cell migration and invasion processes in three-dimensional (3D) extracellular matrixes. However, the information related to the initial stage of cell migration with single-cell resolution, which plays a unique role in the metastasis-invasion cascade of cancer, is not yet available. In this paper, we present a microfluidic device integrated with ECIS for investigating single cancer cell migration in 3D matrixes. Using microfluidics techniques without the requirement of physical connections to off-chip pneumatics, the proposed sensor chip can efficiently capture single cells on microelectrode arrays for sequential on-chip 2D or 3D cell culture and impedance measurement. An on-chip single-cell migration assay was successfully demonstrated within several minutes. Migration of single metastatic MDA-MB-231 cells in their initial stage can be monitored in real time; it shows a rapid change in impedance magnitude of approximately 10 Ω/s, whereas no prominent impedance change is observed for less-metastasis MCF-7 cells. The proposed sensor chip, allowing for a rapid and selective detection of the migratory properties of cancer cells at the single-cell level, could be applied as a new tool for cancer research.