Detection of Breast Cancer-Specific Extracellular Vesicles with Fiber-Optic SPR Biosensor.

Extracellular vesicles (EVs) have attracted great attention as potential biomarkers for cancer diagnostics. Although several technologies have been developed for EV detection, many of them are still not applicable to clinical settings as they rely on complex EV isolation processes, while lacking sensitivity, specificity or standardization. To solve this problem, we have developed a sensitive breast cancer-specific EV detection bioassay directly in blood plasma using a fiber-optic surface plasmon resonance (FO-SPR) biosensor, previously calibrated with recombinant EVs. First, we established a sandwich bioassay to detect SK-BR-3 EVs by functionalizing the FO-SPR probes with anti-HER2 antibodies. A calibration curve was built using an anti-HER2/Banti-CD9 combination, resulting in an LOD of 2.1 × 107 particles/mL in buffer and 7 × 108 particles/mL in blood plasma. Next, we investigated the potential of the bioassay to detect MCF7 EVs in blood plasma using an anti-EpCAM/Banti-mix combination, obtaining an LOD of 1.1 × 10 8 particles/mL. Finally, the specificity of the bioassay was proven by the absence of signal when testing plasma samples from 10 healthy people unknown to be diagnosed with breast cancer. The remarkable sensitivity and specificity of the developed sandwich bioassay together with the advantages of the standardized FO-SPR biosensor highlight outstanding potential for the future of EV analysis.

Dielectrophoretic characterization and separation of monocytes and macrophages using 3D carbon-electrodes.

Monocyte heterogeneity and its prevalence are revealed as indicator of several human diseases ranking from cardiovascular diseases to rheumatoid arthritis, chronic kidney diseases, autoimmune multiple sclerosis, and stroke injuries. When monocytes and macrophages are characterized and isolated with preserved genetic, phenotypic and functional properties, they can be used as label-free biomarkers for precise diagnostics and treatment of various diseases. Here, the dielectrophoretic responses of the monocytes and macrophages were examined. We present 3D carbon-electrode dielectrophoresis (carbon-DEP) as a separation tool for U937 monocytes and U937 monocyte-differentiated macrophages. The carbon-electrodes advanced the usability and throughput of DEP separation, presented wider electrochemical stability. Using the 3D carbon-DEP chip, we first identified the selective positive and negative DEP responses and specific crossover frequencies of monocytes and macrophages as their signatures for separation. The crossover frequency of monocytes and macrophages was 17 and 30 kHz, respectively. Next, we separated monocyte and macrophage subpopulations using their specific dielectrophoretic responses. Afterward, we used a fluorescence-activated cell sorter to confirm our results. Finally, we enriched 70% of monocyte cells from the mixed cell population, in other words, concentration of monocyte cells to macrophage cells was five times increased, using the 30-kHz, 10-Vpp electric field and 1 µL/min flow rate.

Dielectrophoretic Separation of Live and Dead Monocytes Using 3D Carbon-Electrodes.

Blood has been the most reliable body fluid commonly used for the diagnosis of diseases. Although there have been promising investigations for the development of novel lab-on-a-chip devices to utilize other body fluids such as urine and sweat samples in diagnosis, their stability remains a problem that limits the reliability and accuracy of readouts. Hence, accurate and quantitative separation and characterization of blood cells are still crucial. The first step in achieving high-resolution characteristics for specific cell subpopulations from the whole blood is the isolation of pure cell populations from a mixture of cell suspensions. Second, live cells need to be purified from dead cells; otherwise, dead cells might introduce biases in the measurements. In addition, the separation and characterization methods being used must preserve the genetic and phenotypic properties of the cells. Among the characterization and separation approaches, dielectrophoresis (DEP) is one of the oldest and most efficient label-free quantification methods, which directly purifies and characterizes cells using their intrinsic, physical properties. In this study, we present the dielectrophoretic separation and characterization of live and dead monocytes using 3D carbon-electrodes. Our approach successfully removed the dead monocytes while preserving the viability of the live monocytes. Therefore, when blood analyses and disease diagnosis are performed with enriched, live monocyte populations, this approach will reduce the dead-cell contamination risk and achieve more reliable and accurate test results.

FO-SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices.

Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well-characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO-SPR) bioassay. In this context, EV binding on the FO-SPR probes was achieved only with EV-specific antibodies (e.g. anti-CD9 and anti-CD63) but not with non-specific anti-IgG. To increase detection sensitivity, we tested six different combinations of EV-specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti-CD9/Banti-CD81 and anti-CD63/Banti-CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti-CD63/Banti-CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti-EpCAM antibody on the FO-SPR surface. The obtained results combined with FO-SPR real-time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis.

Single Cell Level Dielectrophoretic Responses & Dielectrophoretic Deformations of Monocytes to Quantify Population Heterogeneity.

Single-cell dielectrophoretic movement and dielectrophoretic deformation of monocyte cells were interrogated applying 20 Vpp, 50 kHz to 1 MHz signal in the 3D carbon electrode array. Heterogeneity of the monocyte population is shown in terms of the crossover frequencies, translational movement, and deformation index of the cells. The results presented that crossover range for monocytes was 100 kHz - 200 kHz, the translational movement of the cells was rapidly altered when the initial positions of the cells were in the negative dielectrophoretic region. Finally, the deformation index of the monocyte population varied from 0.5 to 1.5.

Quantitative Investigation for the Dielectrophoretic Effect of Fluorescent Dyes at Single-Cell Resolution.

Most of the microscopy-based, quantitative assays rely on fluorescent dyes. In this study, we investigated the impact of fluorescent dyes on the dielectrophoretic response of the mammalian cells. The dielectrophoretic measurements were performed to quantify whether the fluorescent dyes alter the dielectrophoretic properties of the cells at single-cell resolution. Our results present that when 10 Vpp electric field is applied, the fluorescent-labeled cells experienced the crossover frequency at 8-10 kHz, whereas the label-free cells exhibited at 16-18 kHz.