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Circulating cell free DNA (cfDNA) is a valuable source of biomarkers for a range of medical applications including detection and monitoring of diseases. Currently, cfDNA sequence analysis must take place in a laboratory setting, due to the multiple steps required for processing including collection, purification, amplification, and analysis. Developing a point-of-care test system that combines these steps would simplify DNA processing thereby increasing diagnostic screening accessibility and enabling real-time monitoring for individual patients. Here, we have developed a system that combines multiple cfDNA processing steps into a single microfluidic-based device. This includes cfDNA collection directly from undiluted human plasma followed by purification and on chip amplification. A microelectrode array embedded within the microfluidic chip collected cfDNA through the creation of dielectrophoretic (DEP) forces. DEP utilizes differences in dielectric properties between cfDNA and plasma to preferentially induce a force on cfDNA. We then achieved on-chip amplification of collected DNA by designing a thermal cycling system to enable polymerase chain reaction (PCR) on the chip. This successfully consolidated the most labor-intensive steps of collection, purification, and amplification into a single device. Compared to elution of cfDNA for off-chip amplification, our on-chip PCR method improved the lower limit of detection by 3-fold and improved the total DNA yield by 5-fold. Furthermore, we demonstrate its clinical diagnostic potential by detecting KRAS mutations from a pancreatic ductal adenocarcinoma patient using only 60 μL of plasma. This paves the way for future development of a fully self-contained system facilitating the rapid detection of mutations in cfDNA., Competing Interests: Conflicts of Interest: M. Heller is on the scientific advisory board of Biological Dynamics.

We present a follow-on technique for the cyclic-immunofluorescence profiling of suspension particles isolated using dielectrophoresis. The original lab-on-chip technique ("cyc-DEP" [cyclic immunofluorescent imaging on dielectrophoretic chip]) was designed for the multiplex surveillance of circulating biomarkers. Nanoparticles were collected from low-volume liquid biopsies using microfluidic dielectrophoretic chip technology. Subsequent rounds of cyclic immunofluorescent labeling and quenching were imaged and quantified with a custom algorithm to detect multiple proteins. While cyc-DEP improved assay multiplicity, long runtimes threatened its clinical adoption. Here, we modify the original cyc-DEP platform to reduce assay runtimes. Nanoparticles were formulated from human prostate adenocarcinoma cells and collected using dielectrophoresis. Three proteins were labeled on-chip with a mixture of short oligonucleotide-conjugated antibodies. The sample was then incubated with complementary fluorophore-conjugated oligonucleotides, which were dehybridized using an ethylene carbonate buffer after each round of imaging. Oligonucleotide removal exhibited an average quenching efficiency of 98 ± 3% (n = 12 quenching events), matching the original cyc-DEP platform. The presented "oligo cyc-DEP" platform achieved clinically relevant sample-to-answer times, reducing the duration for three rounds of cyclic immunolabeling from approximately 20 to 6.5 h-a 67% decrease attributed to rapid fluorophore removal and the consolidated co-incubation of antibodies., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies., (© 2022 Wiley-VCH GmbH.)

The 20th century has seen tremendous innovation of dielectrophoresis (DEP) technologies, with applications being developed in areas ranging from industrial processing to micro- and nanoscale biotechnology. From 2010 to present day, there have been 981 publications about DEP. Of over 2600 DEP patents held by the United States Patent and Trademark Office, 106 were filed in 2019 alone. This review focuses on DEP-based technologies and application developments between 2010 and 2020, with an aim to highlight the progress and to identify potential areas for future research. A major trend over the last 10 years has been the use of DEP techniques for biological and clinical applications. It has been used in various forms on a diverse array of biologically derived molecules and particles to manipulate and study them including proteins, exosomes, bacteria, yeast, stem cells, cancer cells, and blood cells. DEP has also been used to manipulate nano- and micron-sized particles in order to fabricate different structures. The next 10 years are likely to see the increase in DEP-related patent applications begin to result in a greater level of technology commercialization. Also during this time, innovations in DEP technology will likely be leveraged to continue the existing trend to further biological and medical-focused applications as well as applications in microfabrication. As a tool leveraged by engineering and imaginative scientific design, DEP offers unique capabilities to manipulate small particles in precise ways that can help solve problems and enable scientific inquiry that cannot be addressed using conventional methods., (© 2021 The Authors. Electrophoresis published by Wiley-VCH GmbH.)

Background: Technology platforms that afford biomarker discovery in patients suffering from traumatic brain injury (TBI) remain an unmet medical need. Here, we describe an observational pilot study to explore the utility of an alternating current electrokinetic (ACE) microchip device in this context. Methods: Blood samples were collected from participating subjects with and without minor TBI. Plasma levels of glial fibrillary acidic protein (GFAP), Tau, ubiquitin C-terminal hydrolase L1 (UCH-L1), and cell-free DNA (cfDNA) were determined in subjects with and without minor TBI using ACE microchip device followed by on-chip immunofluorescent analysis. Post-concussive symptoms were assessed using the Rivermead Post Concussion Symptoms Questionnaire (RPCSQ) at one-month follow-up. Results: Highest levels of GFAP, UCH-L1, and Tau were seen in two minor TBI subjects with abnormality on head computed tomography (CT). In patients without abnormal head CT, Tau and GFAP levels discriminated between plasma from minor-TBI and non-TBI patients, with sensitivity and specificity of 64-72 and 50%, respectively. Plasma GFAP, UCH-L1, and Tau strongly correlated with the cumulative RPCSQ score. Plasma UCH-L1 and GFAP exhibited highest correlation to sensitivity to noise and light ( r = 0.96 and 0.91, respectively, p < 0.001). Plasma UCH-L1 and Tau showed highest correlation with headache ( r = 0.74 and 0.78, respectively, p < 0.001), sleep disturbance ( r = 0.69 and 0.84, respectively, p < 0.001), and cognitive symptoms, including forgetfulness ( r = 0.76 and 0.74, respectively, p < 0.001), poor concentration ( r = 0.68 and 0.76, respectively, p < 0.001), and time required for information processing ( r = 0.77 and 0.81, respectively, p < 0.001). cfDNA exhibited a strong correlation with depression ( r = 0.79, p < 0.01) and dizziness ( r = 0.69, p < 0.01). While cfDNA demonstrated positive correlation with dizziness and depression ( r = 0.69 and 0.79, respectively, p < 0.001), no significant correlation was observed between cumulative RPCSQ and cfDNA ( r = 0.07, p = 0.81). Conclusion: We provide proof-of-principle results supporting the utility of ACE microchip for plasma biomarker analysis in patients with minor TBI., (Copyright © 2020 Lewis, Dhawan, Obirieze, Sarno, Akers, Heller and Chen.)

Objective : To examine resting and postprandial peripheral protease activity in healthy controls and individuals with type 2 diabetes mellitus (T2DM) and pre-T2DM. Methods : Individuals with T2DM or pre-T2DM and healthy controls (mean age 55.8 years) were studied before and for a span of 300 minutes following a single high-calorie McDonald's breakfast. Metalloproteases-2/-9 (MMP-2/-9), elastase, and trypsin activities were assessed in whole blood before and following the meal using a novel high-precision electrophoretic platform. Also assessed were circulating levels of inflammatory biomarkers and insulin receptor density on peripheral blood mononuclear cells (PBMCs) in relationship to protease activity. Results : Premeal MMP-2/-9 and elastase activity levels in T2DM and in pre-T2DM participants were significantly elevated as compared to controls. The T2DM group showed a significant increase in elastase activity 15 minutes after the meal; elastase activity continued to increase to the 30-minute time point ( p  < 0.01). In control participants, MMP-2/-9, elastase, and trypsin were significantly increased at 15 minutes after the meal ( p  < 0.05) and returned to premeal values within a period of approximately 30 to 60 minutes post meal. PBMCs incubated for 1 hour with plasma from T2DM and pre-T2DM participants had significantly lower levels of insulin receptor density compared to those incubated with plasma from control participants ( p  < 0.001). Conclusions : The results of this study suggest that individuals with T2DM and pre-T2DM have higher resting systemic protease activity than nonsymptomatic controls. A single high-calorie/high-carbohydrate meal results in further elevations of protease activity in the systemic circulation of T2DM and pre-T2DM, as well as in healthy controls. The protease activity in turn can lead to a downregulation of insulin receptor density, potentially supporting a state of insulin resistance.

Pancreatic ductal adenocarcinoma (PDAC) typically has nonspecific symptoms and is often found too late to treat. Because diagnosis of PDAC involves complex, invasive, and expensive procedures, screening populations at increased risk will depend on developing rapid, sensitive, specific, and cost-effective tests. Exosomes, which are nanoscale vesicles shed into blood from tumors, have come into focus as valuable entities for noninvasive liquid biopsy diagnostics. However, rapid capture and analysis of exosomes with their protein and other biomarkers have proven difficult. Here, we present a simple method integrating capture and analysis of exosomes and other extracellular vesicles directly from whole blood, plasma, or serum onto an AC electrokinetic microarray chip. In this process, no pretreatment or dilution of sample is required, nor is it necessary to use capture antibodies or other affinity techniques. Subsequent on-chip immunofluorescence analysis permits specific identification and quantification of target biomarkers within as little as 30 min total time. In this initial validation study, the biomarkers glypican-1 and CD63 were found to reflect the presence of PDAC and thus were used to develop a bivariate model for detecting PDAC. Twenty PDAC patient samples could be distinguished from 11 healthy subjects with 99% sensitivity and 82% specificity. In a smaller group of colon cancer patient samples, elevated glypican-1 was observed for metastatic but not for nonmetastatic disease. The speed and simplicity of ACE exosome capture and on-chip biomarker detection, combined with the ability to use whole blood, will enable seamless "sample-to-answer" liquid biopsy screening and improve early stage cancer diagnostics.

DNA has been employed to either store digital information or to perform parallel molecular computing. Relatively unexplored is the ability to combine DNA-based memory and logical operations in a single platform. Here, we show a DNA tri-level cell non-volatile memory system capable of parallel random-access writing of memory and bit shifting operations. A microchip with an array of individually addressable electrodes was employed to enable random access of the memory cells using electric fields. Three segments on a DNA template molecule were used to encode three data bits. Rapid writing of data bits was enabled by electric field-induced hybridization of fluorescently labeled complementary probes and the data bits were read by fluorescence imaging. We demonstrated the rapid parallel writing and reading of 8 (2 3 ) combinations of 3-bit memory data and bit shifting operations by electric field-induced strand displacement. Our system may find potential applications in DNA-based memory and computations.

Recently, instead of indium tin oxide, the random mesh pattern of metallic nanowires for flexible transparent conducting electrodes (FTCEs) has received a great amount of interest due to its flexibility, low resistance, reasonable price, and compliant processes. Mostly, nanowires for FTCEs are fabricated by spray or mayer coating methods. However, the metallic nanowire layer of FTCEs, which is fabricated by these methods, has a spiked surface roughness and low junction contact between the nanowires that lead to their high sheet resistance value. Also, the nanowires on FTCEs are easy to peel-off through exterior forces such as bending, twisting, or contact. To solve these problems, we demonstrate novel methods through which silver nanowires (AgNWs) are deposited onto a nanosize porous nitrocellulose (NC) substrate by electrophoretic deposition (EPD) and an opaque and porous substrate. Respectively, through dimethyl sulfoxide treatment, AgNWs on NC (AgNW/NC) is changed to the transparent and nonporous FTCEs. This enhances the junction contact of the AgNWs by EPD and also allows a permanent attachment of AgNWs onto the substrate. To show the mechanical strength of the AgNWs on the transparent nitrocellulose (AgNW/TNC), it is tested by applying diverse mechanical stress, such as a binding test (3M peel-off), compressing, bending, twisting, and folding. Next, we demonstrate that AgNW/TNC can be effectively implanted onto normal newspapers and papers. As paper electronics, light-emitting diodes, which are laminated onto paper, are successfully operated through a basic AgNW/TNC strip circuit. Finally, it is demonstrated that AgNW/TNC and AgNW/TNC on paper are water resistant for 15 min due to the insulation properties of the nonporous substrate.

A systematic strategy for designing structured nanomaterials is demonstrated through self-assembly of graphene quantum dots. The approach reveals that graphene derivatives at the nanoscale assemble into various architectures of nanocrystals in a binary solution system. The shapes of the nanocrystals continue to evolve in terms of the intimate association of organic molecules with the dispersion medium, obtaining a high index faceted superlattice. This facile synthetic process provides a versatile strategy for designing particles to new structured materials systems, exploiting the crystallization of layered graphitic carbon structures within single crystals., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 μL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

The quest for new cell-free DNA and exosome biomarker-based molecular diagnostics require fast and efficient sample preparation techniques. Conventional methods for isolating these biomarkers from blood are both time-consuming and laborious. New electrokinetic microarray devices using dielectrophoresis (DEP) to isolate cell-free DNA and exosome biomarkers have now greatly improved the sample preparation process. Nevertheless, these devices still have some limitations when used with high conductance biological fluids, e.g. blood, plasma, and serum. This study demonstrates that electrochemical damage may occur on the platinum electrodes of DEP microarray devices. It further examines two model device designs that include a parallel wire arrangement and a planar array. Effective isolation of fluorescent beads with parallel wires is shown under low-conductance conditions (10 -4  S/m), but electrothermal flow overcomes DEP forces under high conductance conditions (>0.1 S/m). Planar devices are shown to be effective under high conductance conditions (∼1 S/m) without the deleterious effects of electrothermal flow. This study provides new insights into design compromises and limitations for producing future electrokinetic devices for better performance with high conductance solutions., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We demonstrate a DNA double-write process that uses UV to pattern a uniquely designed DNA write material, which produces two distinct binding identities for hybridizing two different complementary DNA sequences. The process requires no modification to the DNA by chemical reagents and allows programmed DNA self-assembly and further UV patterning in the UV exposed and nonexposed areas. Multilayered DNA patterning with hybridization of fluorescently labeled complementary DNA sequences, biotin probe/fluorescent streptavidin complexes, and DNA patterns with 500 nm line widths were all demonstrated.

Present coagulation assays fail to detect mild coagulation disorders, while thrombin-generation (TG) assays solve this problem. However, most of them only work with threated blood samples, which makes them labor intensive, time consuming, unreliable, and expensive. We have developed a TG electrophoretic assay that uses a thrombin specific charge-changing fluorescent peptide substrate, electrophoretic separation, and requires a drop of blood. The limit of detection of the assay was 1.97 nM in phosphate buffer saline and 6.82 nM in citrated whole blood. The assay was used to determine the TG in whole blood from healthy volunteers (n = 6, one aspirin user), over 30 min, after the blood was drawn; the TG increased from a baseline level of 2 × 10(6) RFU to 1.2 × 10(13) RFU. The lag time between the blood draw and initial burst of TG was 6 min for the volunteers (n = 5) and 15 min for the aspirin user. Specificity of the assay was evaluated by reacting our substrate with the heparinized blood samples and other proteases. The TG electrophoretic assay was designed and tested in the whole human blood, requiring no sample preparation, 5 μL of blood, 45 min, and it detected differences in coagulation patterns between a volunteer taking aspirin and non-aspirin users., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aim: Circulating cell free (ccf) DNA contains information about mutations affecting chronic lymphocytic leukemia (CLL). The complexity of isolating DNA from plasma inhibits the development of point-of-care diagnostics. Here, we introduce an electrokinetic method that enables rapid recovery of DNA from plasma., Materials & Methods: ccf-DNA was isolated from 25 µl of CLL plasma using dielectrophoresis. The DNA was used for PCR amplification, sequencing and analysis., Results: The ccf-DNA collected from plasma of 5 CLL patients revealed identical mutations to those previously identified by extracting DNA from CLL cells from the same patients., Conclusion: Rapid dielectrophoresis isolation of ccf-DNA directly from plasma provides sufficient amounts of DNA to use for identification of point mutations in genes associated with CLL progression., Competing Interests: Financial & competing interests disclosure The DEP technology used in the study was the result of original research carried out by the MJ Heller laboratory at the UCSD Moores Cancer Center under NIH NCI NanoTumor Center Grant (U54-CA119335). The work was also funded in part by NIH SBIR grant 1R43CA183414-01. Other than MJ Heller who is on the Scientific Advisory Board of Biological Dynamics (La Jolla, CA), no other authors have any relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance was utilized in the production of this manuscript.

Dimerization and resultant quenching of donor and acceptor dyes conjugated on DNA causes loss of fluorescent resonant energy transfer (FRET) efficiency. However, when complexed with surfactants and divalent metal ions, sheathing effects insulate and shield the DNA structures, reducing dimerization and quenching which leads to significant enhancement of FRET efficiency.

While considerable attempts have been made to recreate the high turnover rates of enzymes using synthetic enzyme mimics, most have failed and only a few have produced minimal reaction rates that can barely be considered catalytic. One particular approach we have focused on is the use of short-sequence peptides that contain key catalytic groups in close proximity. In this study, we designed six different peptides and tested their ability to mimic the catalytic mechanism of the cysteine proteases. Acetylation and deacylation by Ellman's Reagent trapping experiments showed the importance of having phenylalanine groups surrounding the catalytic sites in order to provide greater proximity between the cysteine, histidine, and aspartate amino acid R-groups. We have also carried out all-atom molecular dynamics simulations to determine the distance between these catalytic groups and the overall mechanical flexibility of the peptides. We found strong correlations between the magnitude of fluctuations in the Cys-His distance, which determines the flexibility and interactions between the cysteine thiol and histidine imidazole groups, and the deacylation rate. We found that, in general, shorter Cys-His distance fluctuations led to a higher deacylation rate constant, implying that greater confinement of the two residues will allow a higher frequency of the acetyl exchange between the cysteine thiol and histidine imidazole R-groups. This may be the key to future design of peptide structures with molecular mechanical properties that lead to viable enzyme mimics.

The effect of cetyl-trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye-conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye-conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Traditional methods have limitations for the fabrication of micro-ordered batteries for use in a variety of applications including biomedical and healthcare devices. A new micro-nanofabrication method and system that combines deposition, layering, and patterning processes has now been developed for production of micro-ordered energy storage battery devices and materials. Here, we show an electrical field directed (EFD) deposition array device with a 500 μm active assembly area containing 25 microelectrodes. Using EFD assembly, we demonstrated the patterned deposition of graphite mixtures in water-based and solvent-based solutions. The graphite mixture patterns were confirmed by SEM imaging.

The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using an aqueous single reactor arc discharge process with oil-in-water emulsions allows production of 2D multilayered graphenes (MLGs and 3D graphene-based crumpled/sphere-like particles with low levels of defects). The confinement forces to create 3D particles from 2D MLGs are estimated to be 2.5 μN for crumpled particles and 70 μN for spherical hollow particles., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Most dielectrophoretic (DEP) separations of cells, nanoparticles, and other entities are carried out on microelectrode arrays or in microfluidic device formats. Less work has been directed at designing pipette-type formats that would allow dipping into and recovering specific analytes from samples in microtiter plate formats. In order to address this important area, we have fabricated micropipette tip devices containing a 2% agarose gel plug, a buffer chamber, and platinum electrode as the DEP collection device, to be used in combination with separate sample wells that contain a circular gold electrode. We demonstrated that 200 nm fluorescent nanoparticles could be isolated into DEP high-field regions and separated from 10 μm fluorescent microbeads in high conductance buffer (1× PBS) by applying an alternating current at 10 kHz with a peak-to-peak voltage (Vpp) of 160 Vpp. The collected nanoparticles were then transferred to a new buffer solution. We also demonstrated the DEP isolation and separation of genomic DNA (>50 kbps) from the 10 μm microbeads in high conductance buffer (1× PBS) with transfer of collected DNA to another solution., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As we move into the era of individualized cancer treatment, the need for more sophisticated cancer diagnostics has emerged. Cell-free (cf) nucleic acids (cf-DNA, cf-RNA) and other cellular nanoparticulates are now considered important and selective biomarkers. There is great hope that blood-borne cf-nucleic acids can be used for 'liquid biopsies', replacing more invasive tissue biopsies to analyze cancer mutations and monitor therapy. Conventional techniques for cf-nucleic acid biomarker isolation from blood are generally time-consuming, complicated and expensive. They require relatively large blood samples, which must be processed to serum or plasma before isolation of biomarkers can proceed. Such cumbersome sample preparation also limits the widespread use of powerful, downstream genomic analyses, including PCR and DNA sequencing. These limitations also preclude rapid, point-of-care diagnostic applications. Thus, new technologies that allow rapid isolation of biomarkers directly from blood will permit seamless sample-to-answer solutions that enable next-generation point-of-care molecular diagnostics.

Common epifluorescent microscopy lacks the sensitivity to detect low levels of analytes directly in clinical samples, such as drug delivery nanoparticles or disease related DNA biomarkers. Advanced systems such as confocal microscopes may improve detection, but several factors limit their applications. This study now demonstrates that combining an epifluorescent microscope with a dielectrophoretic (DEP) microelectrode array device enables the detection of nanoparticles and DNA biomarkers at clinically relevant levels. Using DEP microarray devices, nanoparticles and DNA biomarkers are rapidly isolated and concentrated onto specific microscopic locations where they are easily detected by epifluorescent microscopy. In this study, 40nm nanoparticles were detected down to 2-3 × 10(3) /ul levels and DNA was detected down to the 200 pg/ml level. The synergy of epifluorescent microscopy and DEP microarray devices provides a new paradigm for DNA biomarker diagnostics and the monitoring of drug delivery nanoparticle concentrations., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 μL of CLL blood and 5 μL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications., Methods: We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced., Results: PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood., Conclusions: Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

The small intestine plays a key role in the pathogenesis of multiple organ failure following circulatory shock. Current results show that reduced perfusion of the small intestine compromises the mucosal epithelial barrier, and the intestinal contents (including pancreatic digestive enzymes and partially digested food) can enter the intestinal wall and transport through the circulation or mesenteric lymph to other organs such as the lung. The extent to which the luminal contents of the small intestine mediate tissue damage in the intestine and lung is poorly understood in shock. Therefore, rats were assigned to three groups: No-hemorrhagic shock (HS) control and HS with or without a flushed intestine. HS was induced by reducing the mean arterial pressure (30 mmHg; 90 min) followed by return of shed blood and observation (3 h). The small intestine and lung were analyzed for hemorrhage, neutrophil accumulation, and cellular membrane protein degradation. After HS, animals with luminal contents had increased neutrophil accumulation, bleeding, and destruction of E-cadherin in the intestine. Serine protease activity was elevated in mesenteric lymph fluid collected from a separate group of animals subjected to intestinal ischemia/reperfusion. Serine protease activity was elevated in the plasma after HS but was detected in lungs only in animals with nonflushed lumens. Despite removal of the luminal contents, lung injury occurred in both groups as determined by elevated neutrophil accumulation, permeability, and lung protein destruction. In conclusion, luminal contents significantly increase intestinal damage during experimental HS, suggesting transport of luminal contents across the intestinal wall should be minimized.

Dielectrophoretic (DEP) microarray devices allow important cellular nanoparticulate biomarkers and virus to be rapidly isolated, concentrated, and detected directly from clinical and biological samples. A variety of submicron nanoparticulate entities including cell free circulating (cfc) DNA, mitochondria, and virus can be isolated into DEP high-field areas on microelectrodes, while blood cells and other micron-size entities become isolated into DEP low-field areas between the microelectrodes. The nanoparticulate entities are held in the DEP high-field areas while cells are washed away along with proteins and other small molecules that are not affected by the DEP electric fields. DEP carried out on 20 μL of whole blood obtained from chronic lymphocytic leukemia patients showed a considerable amount of SYBR Green stained DNA fluorescent material concentrated in the DEP high-field regions. Whole blood obtained from healthy individuals showed little or no fluorescent DNA materials in the DEP high-field regions. Fluorescent T7 bacteriophage virus could be isolated directly from blood samples, and fluorescently stained mitochondria could be isolated from biological buffer samples. Using newer DEP microarray devices, high-molecular-weight DNA could be isolated from serum and detected at levels as low as 8-16 ng/mL., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ability to effectively detect disease-related DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for viable diagnostics and therapy monitoring. A DEP method has been developed which allows the rapid isolation, concentration and detection of DNA and nanoparticles directly from human and rat whole blood. Using a microarray device operating at 20 V peak-to-peak and 10 kHz, a wide range of high molecular weight (HMW)-DNA and nanoparticles were concentrated into high-field regions by positive DEP, while the blood cells were concentrated into the low-field regions by negative DEP. A simple fluidic wash removes the blood cells while the DNA and nanoparticles remain concentrated in the DEP high-field regions where they can be detected by fluorescence. HMW-DNA could be detected at 260 ng/mL, which is a detection level suitable for analysis of disease-related cell-free circulating DNA biomarkers. Fluorescent 40 nm nanoparticles could be detected at 9.5 × 10(9) particles/mL, which is a level suitable for monitoring drug delivery nanoparticles. The ability to rapidly isolate and detect DNA biomarkers and nanoparticles from undiluted whole blood will benefit many diagnostic applications by significantly reducing sample preparation time and complexity., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A method is presented for the electric-field-directed self-assembly of higher-order structures composed of alternating layers of biotin nanoparticles and streptavidin-/avidin-conjugated enzymes carried out on a microelectrode array device. Enzymes included in the study were glucose oxidase (GOx), horseradish peroxidase (HRP), and alkaline phosphatase (AP); all of which could be used to form a light-emitting microscale glucose sensor. Directed assembly included fabricating multilayer structures with 200 nm or 40 nm GOx-avidin-biotin nanoparticles, with AP-streptavidin-biotin nanoparticles, and with HRP-streptavidin-biotin nanoparticles. Multilayered structures were also fabricated with alternate layering of HRP-streptavidin-biotin nanoparticles and GOx-avidin-biotin nanoparticles. Results showed that enzymatic activity was retained after the assembly process, indicating that substrates could still diffuse into the structures and that the electric-field-based fabrication process itself did not cause any significant loss of enzyme activity. These methods provide a solution to overcome the cumbersome passive layer-by-layer assembly methods to efficiently fabricate higher-order active biological and chemical hybrid structures that can be useful for creating novel biosensors and drug delivery nanostructures, as well as for diagnostic applications.

The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 μL of 1× phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases.

The measurement of trypsin activity directly in blood is important for the development of novel diagnostics and for biomedical research. Presently, most degradative enzyme assays require sample preparation, making them time consuming, costly, and less accurate. We recently demonstrated a simple and rapid electrophoretic assay for the measurement of trypsin activity directly in whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target enzyme. This fragment is then rapidly separated from whole blood by electrophoresis and quantified with a fluorescent detector. In this study, we demonstrate that polyanionic poly-L-glutamic acid-doped polyacrylamide gels can focus the fluorescent cleavage product and markedly improve the LODs of the assay. A LOD of 2 pg in 6 microL (0.3 ng/mL) in whole human blood was achieved after a 1-h reaction of enzyme and substrate followed by 10 min of electrophoresis. This is 50- to 200-fold better than the estimated reference levels for trypsin (15-60 ng/mL) in blood. This straightforward technique now allows for the rapid measurement of clinically relevant levels of trypsin activity in microliter volumes of whole blood, providing a useful tool for the development of novel point-of-care diagnostics.

In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin-specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.

We report a method for rapid, electric field directed assembly of high-density protein-conjugated microbead arrays. Photolithography is used to fabricate an array of micron to sub-micron-scale wells in an epoxy-based photoresist on a silicon wafer coated with a thin gold film, which serves as the primary electrode. A thin gasket is used to form a microfluidic chamber between the wafer and a glass coverslip coated with indium-tin oxide, which serves as the counter electrode. Streptavidin-conjugated microbeads suspended in a low conductance buffer are introduced into the chamber and directed into the wells via electrophoresis by applying a series of low voltage electrical pulses across the electrodes. Hundreds of millions of microbeads can be permanently assembled on these arrays in as little as 30 seconds and the process can be monitored in real time using epifluorescence microscopy. The binding of the microbeads to the gold film is robust and occurs through electrochemically induced gold-protein interactions, which allows excess beads to be washed away or recycled. The well and bead sizes are chosen such that only one bead can be captured in each well. Filling efficiencies greater than 99.9% have been demonstrated across wafer-scale arrays with densities as high as 69 million beads per cm(2). Potential applications for this technology include the assembly of DNA arrays for high-throughput genome sequencing and antibody arrays for proteomic studies. Following array assembly, this device may also be used to enhance the concentration-dependent processes of various assays through the accelerated transport of molecules using electric fields.

The separation of nanoparticles from micron size particles in high conductance buffers was achieved using an AC dielectrophoretic (DEP) microarray device with hydrogel over-coated microelectrodes. While nanoparticles could be selectively concentrated into high field regions directly over the platinum microelectrodes, micro-bubbling and electrode darkening was also observed. For similar experiments using un-coated microelectrodes, SEM analysis showed severe erosion of the platinum microelectrodes and fusion of nanoparticles due to the aggressive electrochemistry.

In biomedical research and diagnostics it is a challenge to isolate and detect low levels of nanoparticles and nanoscale biomarkers in blood and other biological samples. While highly sensitive epifluorescent microscope systems are available for ultra low level detection, the isolation of the specific entities from large sample volumes is often the bigger limitation. AC electrokinetic techniques like dielectrophoresis (DEP) offer an attractive mechanism for specifically concentrating nanoparticles into microscopic locations. Unfortunately, DEP requires significant sample dilution thus making the technology unsuitable for biological applications. Using a microelectrode array device, special conditions have been found for the separation of hmw-DNA and nanoparticles under high conductance (ionic strength) conditions. At AC frequencies in the 3000-10 000 Hz range, 10 mum microspheres and human T lymphocytes can be isolated into the DEP low field regions, while hmw-DNA and nanoparticles can be concentrated into microscopic high field regions for subsequent detection using an epifluorescent system., ((c) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electric field directed hybridization was used to produce twenty layer nanostructures composed of DNA derivatized nanoparticles. Using an electronic microarray device, DNA nanoparticles could be directed and concentrated such that rapid and specific hybridization occurs only on the activated sites. Nanoparticle layers were formed within 30 s of activation and twenty layer structures completed in under an hour. Results demonstrate a unique combination of bottom-up and top-down techniques for nanofabrication.

In biomedical research and diagnostics, it is a significant challenge to directly isolate and identify rare cells and potential biomarkers in blood, plasma and other clinical samples. Additionally, the advent of bionanotechnology is leading to numerous drug delivery approaches that involve encapsulation of drugs and imaging agents within nanoparticles, which now will also have to be identified and separated from blood and plasma. Alternating current (AC) electrokinetic techniques such as dielectrophoresis (DEP) offer a particularly attractive mechanism for the separation of cells and nanoparticles. Unfortunately, present DEP techniques require the dilution of blood/plasma, thus making the technology less suitable for clinical sample preparation. Using array devices with microelectrodes over-coated with porous hydrogel layers, AC electric field conditions have been found which allow the separation of DNA nanoparticles to be achieved under high-conductance (ionic strength) conditions. At AC frequencies in the 3000 Hz to 10,000 Hz range and 10 volts peak-to-peak, the separation of 10-microm polystyrene particles into low field regions, and 60-nm DNA-derivatized nanoparticles and 200-nm nanoparticles into high-field regions was carried out in 149 mM 1xPBS buffer (1.68 S/m). These results may allow AC electrokinetic systems to be developed that can be used with clinically relevant samples under physiological conditions.

Multilayered structures composed of biomolecule-derivatized nanoparticles can be fabricated by electric-field-directed self-assembly. A microelectrode-array device facilitates the rapid parallel electrophoretic transport and binding of biotin and streptavidin fluorescent nanoparticles to specific sites on the microarray. Control of the current, voltage, and activation time of each of the 400-microarray electrodes allows a combinatorial approach to optimize nanoparticle binding. Under optimal conditions, nanoparticle layers form within 15 s of microelectrode activation, and the directed assembly of more than 50 alternate layers of nanoparticles is complete within an hour. The final multilayered structures are removed from the support by a relatively simple lift-off process. The electric-field process allows the parallel patterned assembly of multilayer structures using extremely low concentrations of nanoparticles and produces minimal nonspecific binding to unactivated sites. These results are significant for the development of rapid, maskless nanofabrication and hierarchical integration of biomolecular-derivatized nanocomponents into higher-order materials and devices.

Inherent bistabilities within DNA-assembled fluorescent resonant energy transfer systems demonstrated time-varying optical signals in response to an electrophoretic driving force. Frequency responses of electrophoretically driven FRET systems were shown to be sequence specific. Integration of these signals over time gave near single-molecule sensitivity within a high background of autofluorescence. This research suggests that externally driven nanoscale mechanical systems may help improve information flow within morphologically intact specimens.

Is hospitalization needed for patients in whom an underlying cause of syncope is not detected in the Emergency Department (ED)? To answer this question, we conducted a prospective, short-term outcomes study of consecutive patients > or = 50 years old presenting to our ED after a syncopal episode. All subjects received a structured ED evaluation. Forty-five patients met inclusion criteria, 67% were hospitalized. None of the patients experienced a life-threatening event or required significant therapeutic interventions during the hospitalization. No patient had a new diagnosis relevant to syncope. Follow-up interviews 1 month later revealed no repeat ED visits, hospitalizations, or deaths (95% upper CI, 6.5%). One patient reported a recurrent syncope (recurrence 2.2% [95% upper CI, 10%]). This pilot study suggests that a negative structured ED evaluation may identify patients > or = 50 years of age who may be safely discharged from the ED. Further validation is required before this observation is applied to clinical practice.

A technique of detecting the rolling and spinning of half-coated nanoparticles using interference ring patterns of the fluorescence has been applied to the measurement of torsional spring modulus of a double-stranded DNA. Using the unique ability to measure nanoparticle rotations in multiple degrees of freedom, we were able to determine the spinning of a nanoparticle tethered on the DNA and thereby the twisting of the DNA in real time. The detailed knowledge of the spinning as well as rolling behaviors of half-coated nanoparticles provides information about torsional elastic properties of the DNA under investigation.

The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of alpha-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10 degrees C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 degrees C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0-1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca2+ binding sites of thin filament in two settings: Ca2+-dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca2+ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca2+ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca2+ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca2+ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.

Microelectronic arrays have been developed for DNA hybridization analysis of point mutations, single nucleotide polymorphisms, short tandem repeats and gene expression. In addition to a variety of molecular biology and genomic research applications, such devices will also be used for infectious disease detection, genetic and cancer diagnostics, and pharmacogenomic applications. These microelectronic array devices are able to produce defined electric fields on their surfaces that allow charged molecules and other entities to be transported to or from any test site or micro-location on the planar surface of the device. These molecules and entities include DNA, RNA, proteins, enzymes, antibodies and cells. Electronic-based molecule addressing and hybridization can then be carried out, where the electric field is now used to greatly accelerate the hybridization reactions that occur on the selected test sites. When reversed, the electric field can be used to provide an additional parameter for improved hybridization. Special low-conductance buffers have been developed that provide for the rapid transport of the DNA molecules and facilitate the electronic hybridization reactions under conditions that do not support hybridization. Important to the device function is the permeation layer that overcoats the underlying microelectrodes. Generally composed of a porous hydrogel material impregnated with attachment chemistry, this permeation layer prevents the destruction of analytes at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization and affinity reactions, and serves as a support structure for attaching DNA probes and other molecules to the array. The microelectronic chip or array device is incorporated into a cartridge package (NanoChip trade mark cartridge) that provides the electronic, optical, and fluidic interfacing. A complete instrument system (NanoChip trade mark Molecular Biology Workstation) provides a chip loader, fluorescent reader, computer control interface and data display screen. The probe loader component allows DNA probes or target molecules (polymerase chain reactions amplicons, genomic DNA, RNA, etc.) to be selectively addressed to the array test sites, providing the end-user with 'make your own chip' capabilities. The electronic hybridization can then be carried out and the chip analyzed using a fluorescent detector system. In addition to carrying out rapid, accurate and highly reliable genotyping (point mutations, single nucleotide polymorphisms, short tandem repeats), other future applications include gene expression analysis, or on-chip amplification, immunoassays and cell separation and selection. Smaller and more compact systems are also being designed for portable sample to answer and point of care diagnostics.

A variety of electronic DNA array devices and techniques have been developed that allow electric field enhanced hybridization to be carried out under special low-conductance conditions. These devices include both planar microelectronic DNA array/chip devices as well as electronic microtiter plate-like devices. Such "active" electronic devices are able to provide controlled electric (electrophoretic) fields that serve as a driving force to move and concentrate nucleic acid molecules (DNA/RNA) to selected microlocation test-sites on the device. In addition to ionic strength, pH, temperature and other agents, the electric field provides another controllable parameter that can affect and enhance DNA hybridization. With regard to the planar microelectronic array devices, special low-conductance buffers were developed in order to maintain rapid transport of DNA molecules and to facilitate hybridization within the constrained low current and voltage ranges for this type of device. With regard to electronic microtiter plate type devices (which do not have the low current/voltage constraints), the use of mixed buffers (low conductance upper chamber/high conductance lower chamber) can be used in a unique fashion to create favorable hybridization conditions in a microzone within the test site location. Both types of devices allow DNA molecules to be rapidly and selectively hybridized at the array test sites under conditions where the DNA in the bulk solution can remain substantially denatured.

In this review, recent advances in DNA microarray technology and their applications are examined. The many varieties of DNA microarray or DNA chip devices and systems are described along with their methods for fabrication and their use. This includes both high-density microarrays for high-throughput screening applications and lower-density microarrays for various diagnostic applications. The methods for microarray fabrication that are reviewed include various inkjet and microjet deposition or spotting technologies and processes, in situ or on-chip photolithographic oligonucleotide synthesis processes, and electronic DNA probe addressing processes. The DNA microarray hybridization applications reviewed include the important areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). In addition to the many molecular biological and genomic research uses, this review covers applications of microarray devices and systems for pharmacogenomic research and drug discovery, infectious and genetic disease and cancer diagnostics, and forensic and genetic identification purposes. Additionally, microarray technology being developed and applied to new areas of proteomic and cellular analysis are reviewed.

Bioparticle separation, bioparticle enrichment, and electric field-mediated immune detection were carried out on microfabricated semiconductor chips utilizing ac and dc electric fields. Microscale separation on a chip surface having an active area of approximately 16 mm2 was demonstrated for a mixture of Bacillus globigii spores and Escherichia coli bacteria. Dielectrophoretic enrichment was performed by collecting target bioparticles from a flow stream in flow cells of 47.5 microL, achieving a 20-fold increase in the concentration of E. coli bacteria from a diluted sample, a 28-fold enrichment for peripheral blood mononuclear cells from red blood cells, and a 30-fold increase in white blood cells from diluted whole blood. The ability to manipulate and collect bioparticles and macromolecules at microfabricated electrodes with ac and dc fields was further illustrated in electric field-mediated immunoassays for analyzing the biological identities of E. coli bacteria and B. globigii spores. According to these results, the electric methods for manipulating bioparticles present themselves as viable techniques for novel biomedical applications in sample preparations and biochemical assays on microelectrode arrays.

Microelectronic DNA chip devices that contain planar arrays of microelectrodes have been developed for multiplex DNA hybridization and a variety of genomic research and DNA diagnostic applications. These devices are able to produce almost any desired electric field configuration on their surface. This ability to produce well-defined electric fields allows charged molecules (DNA, RNA, proteins, enzymes, antibodies, nanobeads, and even micron scale semiconductor devices) to be electrophoretically transported to or from any microlocation on the planar surface of the device. Of key importance to the device function is the permeation layer which overcoats the microelectrodes. The permeation layer is generally a porous hydrogel material that allows water molecules and small ions (Na+, CI-, etc.) to freely contact the microelectrode surface, but impedes the transport of the larger analytes (oligonucleotides, DNA, RNA, proteins, etc.). The permeation layer prevents the destruction of DNA at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization reactions, and serves as a porous support structure for attaching DNA probes and other molecules to the array. In order to maintain rapid transport of DNA molecules, facilitate hybridization, and work within constrained current and voltage ranges, low conductance buffers and various electronic pulsing scenarios have also been developed. These active microelectronic array devices allow electrophoretic fields to be used to carry out accelerated DNA hybridization reactions and to improve selectivity for single nucleotide polymorphism (SNP), short tandem repeat (STR), and point mutation analysis.

Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

The separation and subsequent isolation of the metastatic human cervical carcinoma cell line (HeLa cells) from normal human peripheral blood cells has been achieved by exploiting their differential dielectric properties. The isolation process is carried out on a silicon chip containing a five-by-five array of microlocations. These microlocations contain underlying circular platinum electrodes with 80-micron diameters and center-to-center spacing of 200 microns. The surfaces of the electrodes and nonmetallized areas have been coated with a permeation layer to prevent the direct contact of cells with the electrode and also to minimize the nonspecific adhesion of the cells to the chip surface. An inhomogenous ac field is applied to the electrodes to create the conditions for dielectrophoretic separation of cells. Cell separation using dielectrophoresis as well as electronic lysis on a silicon chip would provide essential sample-processing steps which may be combined with a later multiplex electronic hybridization step in an integrated assay system.