Cell membrane dielectric properties of five different cultivated cell lines and human peripheral blood mononuclear cells (PBMC) were determined from dielectrophoretic crossover frequency measurements on a 5 x 5 microelectronic chip array. Based on distinct dielectric property differences between individual cell types, efficient cell separations were achieved by dielectrophoresis on this 5 x 5 array, which included separation of monocytic cells (U937) or human T cell leukemia virus type 1 (HTLV-1) tax-transformed cells (Ind-2) from PBMC, as well as separation of neuroblastoma cells (SH-SY5Y) from glioma cells (HTB). The purity of dielectrophoretically separated cells can be greater than 95%. Expression profiles of IL-1, TNF-alpha, and TGF-beta genes for U937 cells mixed with PBMC before and after the separation were determined by a means of electric field-facilitated hybridization on a 10 x 10 microelectronic chip array. By using the expression levels of pure U937 cells as a control, it was shown that the gene expression profiles of the postseparation cells were significantly different from those of the preseparation cell mixtures. The increase in gene expression levels for U937 cells upon lipopolysaccharide induction could be accurately determined only in the postseparation cells, while the preseparation samples masked these changes. Furthermore, by cultivating the separated HTB and SH-SY5Y cells and measuring expression of the stress-related gene c-fos, dielectrophoretic forces were shown to have little effect on cell survival and stress. The presented approach of using microelectronic chip arrays for both cell separation and gene expression profiling provides a great potential for accurate genetic analysis of specific cell subpopulations in heterogeneous samples.

Dielectrophoretic cell separation and gene expression profiling on microelectronic chip arrays

▪ Nacido en Cartagena (Murcia), en noviembre de 1954. ▪ Licenciado en Ciencias Químicas por la Universidad de Murcia, Catedrático de Física y Química, Divulgador Científico freelance, Escritor, y Conferenciante. ▪ Con 35 años de experiencia docente, ejerciendo como profesor. ▪ A lo largo de su dilatada carrera docente, ha desempeñado cargos de responsabilidad en la gestión de Centros Escolares, tales como Director (7 años), Jefe de Estudios (2 años), Jefe de Departamento de Física y Química (20 años), miembro del Consejo Escolar de Instituto y miembro del Consejo del Centro de Profesores y Recursos de Cartagena (11 años). ▪ Presentación de comunicaciones y ponencias en 28 Congresos, Jornadas y Seminarios. ▪ Ha sido durante 13 años tutor del segundo ciclo del curso de Aptitud Pedagógica de Educación Secundaria, organizado por el Instituto de Ciencias de la Educación, de la Universidad de Murcia y la Universidad Complutense de Madrid. ▪ También ha sido, durante 1 año, tutor de la fase de prácticas del Máster de Profesor de Educación Secundaria, organizado por el Instituto de Ciencias de la Educación, de la Universidad de Murcia.

Curriculum Vitae for

The complexity of biological analytes demands powerful analysis techniques in order to reveal cellular processes and pathways on the molecular level.1 Thus, ever refining analytical tools or combinations of various analysis techniques are required to reveal such complexities. Dielectrophoresis (DEP) refers to an analytical technique in which a polarizable particle experiences an attractive or repulsive force when placed in a non-uniform electric field. Realized in microenvironments, DEP phenomena have allowed innovative analytical applications due to the strong dependency on the size of the analyte in conjunction with its dielectric properties and are especially suitable for manipulating µm-sized, cellular bioanalytes. However, the demonstration of DEP for smaller sub-cellular entities or non-biological, sub-µm sized particles has been more difficult, as the required DEP forces impose design challenges.2 In addition, the structural complexity of sub-cellular bioanalytes, and the huge variety of microbes s...

Dielectrophoresis: From Molecular to Micrometer-Scale Analytes.

Biosensors for early diagnosis of pancreatic cancer: a review

Exploitation of the intrinsic electrical properties of particles has recently emerged as an appealing approach for trapping and separating various scaled particles. Initiative particle manipulation by dielectrophoresis (DEP) showed remarkable advantages including high speed, ease of handling, high precision and being label-free. Herein, we provide a general overview of the manipulation of polystyrene (PS) beads and related particles via DEP; especially, the wide applications of these manipulated PS beads in the quantitative evaluation of device performance for model validation and standardization have been discussed. The motion and polarizability of the PS beads induced by DEP were analyzed and classified into two categories as positive and negative DEP within the time and space domains. The DEP techniques used for bioparticle manipulation were demonstrated, and their applications were conducted in four fields: trapping of single-sized PS beads, separation of multiple-sized PS beads by size, separation of PS beads and non-bioparticles, and separation of PS beads and bioparticles. Finally, future perspectives on DEP-on-a-chip have been proposed to discriminate bio-targets in the network of microfluidic channels.

/pdf/a-review-of-polystyrene-bead-manipulation-by-2832weiase.pdf

A review of polystyrene bead manipulation by dielectrophoresis

The current gold standard for detecting or quantifying target analytes from blood samples is the ELISA (enzyme-linked immunosorbent assay). The detection limit of ELISA is about 250 pg/ml. However, to quantify analytes that are related to various stages of tumors including early detection requires detecting well below the current limit of the ELISA test. For example, Interleukin 6 (IL-6) levels of early oral cancer patients are 100 pg/ml and the prostate specific antigen level of the early stage of prostate cancer is about 1 ng/ml. Further, it has been reported that there are significantly less than $1\phantom{\rule{0.16em}{0ex}}\mathrm{pg}/\mathrm{mL}$ of analytes in the early stage of tumors. Therefore, depending on the tumor type and the stage of the tumors, it is required to quantify various levels of analytes ranging from ng/ml to pg/ml. To accommodate these critical needs in the current diagnosis, there is a need for a technique that has a large dynamic range with an ability to detect extremely low levels of target analytes (/ml). To address this gap, we here report on a label-free, high-throughput technique based on dielectrophoresis. This technique is capable of quantifying target analytes down to a few thousands of molecules ($\ensuremath{\sim}\mathrm{zmoles}$).

Dielectrophoretic label-free immunoassay for rare-analyte quantification in biological samples.

iLluminate-miRNA: Paradigm for high-throughput, low-cost, and sensitive miRNA detection in serum samples at point-of-care

We propose the use of negative dielectrophoresis (DEP) spectroscopy as a technique to improve the detection limit of rare analytes in biological samples. We observe a significant dependence of the negative DEP force on functionalized polystyrene beads at the edges of interdigitated electrodes with respect to the frequency of the electric field. We measured this velocity of repulsion for 0% and 0.8% conjugation of avidin with biotin functionalized polystyrene beads with our automated software through real-time image processing that monitors the Rayleigh scattering from the beads. A significant difference in the velocity of the beads was observed in the presence of as little as 80 molecules of avidin per biotin functionalized bead. This technology can be applied in the detection and quantification of rare analytes that can be useful in the diagnosis and the treatment of diseases, such as cancer and myocardial infarction, with the use of polystyrene beads functionalized with antibodies for the target biomarkers.

https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-22/issue-3/037006/Negative-dielectrophoresis-spectroscopy-for-rare-analyte-quantification-in-biological-samples/10.1117/1.JBO.22.3.037006.pdf

Negative dielectrophoresis spectroscopy for rare analyte quantification in biological samples.

We present an integrated dielectrophoretic (DEP) and surface plasmonic technique to quantify ∼1 pM of fluorescent molecules in low conductivity buffers. We have established a DEP force on target molecules to bring those molecules and place them on the nanometallic structures (hotspots) for quantification through surface plasmonic effects. Our results show that the DEP is capable of placing the fluorescent molecules on the hotspots, which are depicted as a significant reduction in the fluorescence lifetime of those molecules. To efficiently integrate the DEP and plasmonic effects, we have designed and utilized pearl-shaped interdigitated electrodes (PIDEs) in experiments. These electrodes generate 2-3 times higher DEP force than traditional interdigitated electrodes. Therefore, high-throughput assays can be developed. The nanometallic structures were strategically fabricated in the periphery of PIDEs for smooth integration of DEP and plasmonic detection. With the introduction of DEP, about 106-fold improvement was achieved over existing plasmonic-based detection. Therefore, this simple addition to the existing surface plasmonic-based detection will enable the disease related protein detection.

/pdf/integrated-dielectrophoretic-and-surface-plasmonic-platform-3vn1ijk91m.pdf

Integrated dielectrophoretic and surface plasmonic platform for million-fold improvement in the detection of fluorescent events

We show that negative dielectrophoresis (DEP) spectroscopy is an effective transduction mechanism of a biosensor for the diagnosis and prognosis of pancreatic cancer using the biomarker CA 19-9. A substantial change in the negative DEP force applied to functionalized polystyrene microspheres (PM) was observed with respect to both the concentration level of the pancreatic cancer biomarker CA 19-9 and the frequency of the electric field produced by a pearl shaped interdigitated gold micro-electrode. The velocity of repulsion of a set of PM functionalized to a monoclonal antibody to CA 19-9 was calculated for several concentration cutoff levels of CA 19-9, including 0 U/mL and 37 U/mL, at the frequency range from 0.5 to 2 MHz. The velocity of repulsion of the PM from the electrode was determined using a side illumination and an automated software using a real-time image processing technique that captures the Mie scattering from the PM. Since negative DEP spectroscopy is an effective transduction mechanism for the detection of the cutoff levels of CA 19-9, it has the potential to be used in the early stage diagnosis and in the prognosis of pancreatic cancer.

Logeeshan Velmanickam

Papers

Dielectrophoretic label-free immunoassay for rare-analyte quantification in biological samples.

iLluminate-miRNA: Paradigm for high-throughput, low-cost, and sensitive miRNA detection in serum samples at point-of-care

Negative dielectrophoresis spectroscopy for rare analyte quantification in biological samples.

Integrated dielectrophoretic and surface plasmonic platform for million-fold improvement in the detection of fluorescent events

Label-Free Biosensing Method for the Detection of a Pancreatic Cancer Biomarker Based on Dielectrophoresis Spectroscopy