Electrokinetic device design and constraints for use in high conductance solutions.

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.

Electric manipulation of bioparticles and macromolecules on microfabricated electrodes.

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.

Active microeletronic chip devices which utilize controlled electrophoretic fields for multiplex DNA hybridization and other genomic applications.

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.

Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips.

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.

Isolation of cultured cervical carcinoma cells mixed with peripheral blood cells on a bioelectronic chip.

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.

Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control.

We have demonstrated that controlled electric fields can be used to regulate transport, concentration, hybridization, and denaturation of single- and double-stranded oligonucleotides. Discrimination among oligonucleotide hybrids with widely varying binding strengths may be attained by simple adjustment of the electric field strength. When this approach is used, electric field denaturation control allows single base pair mismatch discrimination to be carried out rapidly (<15 sec) and with high resolution. Electric field denaturation takes place at temperatures well below the melting point of the hybrids, and it may constitute a novel mechanism of DNA denaturation.

Electric field directed nucleic acid hybridization on microchips.

Selection and adjustment of proper physical parameters enables rapid DNA transport, site selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. However, only a subset of buffer species produce both rapid transport, site specific concentration and accelerated hybridization. These buffers include zwitterionic and low conductivity species such as: d- and l-histidine; 1- and 3-methylhistidines; carnosine; imidazole; pyridine; and collidine. In contrast, buffers such as glycine, beta-alanine and gamma-amino-butyric acid (GABA) produce rapid transport and site selective concentration but do not facilitate hybridization. Our results suggest that the ability of these buffers (histidine, etc.) to facilitate hybridization appears linked to their ability to provide electric field concentration of DNA; to buffer acidic conditions present at the anode; and in this process acquire a net positive charge which then shields or diminishes repulsion between the DNA strands, thus promoting hybridization.

In vitro stimulation of human endothelial cells by sera from a subpopulation of high-percentage panel-reactive antibody patients.

We have studied a serum activity that enhances in vitro ICAM-1 expression by human endothelial cells (EC) and report that this activity can be found in approximately 8% of pretransplant serum samples from individuals with a history of high %PRA. Hence, most high %PRA sera lack this activity, and, furthermore, mixing these negative sera does not result in an active serum pool. In patients with active serum, the ICAM-1 enhancing activity is found only sporadically, despite the continuous detection of endothelial-reactive antibodies. Absorption of Ig from a high %PRA serum reduced ICAM-1 enhancing activity, as well as endothelial-reactive antibodies. However, enhancing activity can sometimes be observed in sera that lack detectable endothelial-reactive antibodies, and none of several patient sera with defined MHC class I-specific alloantibodies displayed ICAM-1 enhancing activity. Together, these data suggest that ICAM-1 enhancing activity may not necessarily be mediated by anti-MHC alloantibodies. In addition to influencing this expression, ICAM-1 active patient sera also influence EC expression of VCAM-1 and MHC class I, but not MHC class II molecules, a pattern that is similar to that stimulated by TNF alpha. However, coincubation of EC with active serum plus soluble TNF receptor did not block the endothelial phenotypic changes, despite the ability of the soluble receptor to completely abrogate endothelial changes induced by TNF alpha. IFN gamma also increases endothelial ICAM-1 expression, but has response kinetics different from that of active serum. Interestingly, brief treatment of endothelial cells with IFN gamma greatly increased the amount of IgG bound from the active sera by EC. We conclude that some pretransplant patients occasionally express an activity in their serum that influences EC expression of several adhesion molecules, including ICAM-1, VCAM-1, and MHC class I. This activity may be associated with alloantibodies, but is independent of MHC class I-reactive antibodies, circulating TNF alpha, or IFN gamma. The relevance of a serum-borne component capable of activating EC is discussed.

Oncogene amplification screening by labeled primer multiplex polymerase chain reaction.

This paper describes an improved procedure for rapid detection of amplified genes in fresh or formalin-fixed, paraffin-embedded tissues. Utilizing a multiplex differential polymerase chain reaction with radioactively labeled primers and electrophoresis of the products through thin gels, it is possible to screen for oncogene amplification more rapidly and reproducibly than has been previously demonstrated. This procedure takes advantage of thin vertical gels with external cooling, which allows sharp band resolution. Four separate gels can be electrophoresed at the same time in a single gel box. Because each gel slab contains 10 or more lanes, 40 or more samples can be assayed for gene amplification simultaneously. The entire procedure can be carried out from formalin-fixed, paraffin-embedded tissue to finish in 8 h when combined with a sonication technique for DNA extraction.

Use of thiazole orange homodimer as an alternative to ethidium bromide for DNA detection in agarose gels.

Detection of polymerase chain reaction-amplified DNA fragments is commonly accomplished by visualizing the products in electrophoretic agarose beds with the use of ethidium bromide under ultraviolet light. However, ethidium bromide is mutagenic, and special handling and disposal precautions must be used. We report the use of a nonmutagenic dye, thiazole orange dimer (TOTO), which can be substituted for ethidium bromide. The excitation maximum for TOTO under ultraviolet light is 488 nm, and the absorption maximum is 510 nm, necessitating photographic filters different from those used for ethidium bromide for optimal results. Of particular importance in TOTO's use is the quantity used for each gel lane, since excess TOTO will cause unacceptable product mobility retardation. TOTO is only slightly more expensive than ethidium bromide. Overall, this stain provides very good visualization of polymerase chain reaction--amplified DNA bands in agarose gels. We believe the use of this safer reagent will become more widespread with increased regulation of laboratory activities.

Evaluation of an automated method of percent reactive antibody determination.

A fluorescence-based automated method of percent reactive antibody (PRA) analysis is described. This method utilizes the conventional antibody-mediated, C'-dependent lymphocyte microcytotoxicity assay to detect alloantibodies, but replaces the eosin-based method for detection of cell death with a fluorescence-based method. To identify viable cells, lymphocytes were pretreated with carboxy fluorescein diacetate (CFDA), which fluoresces green, to identify viable cells. To identify dead cells after the reaction with antibody and C', they were treated with propidium iodide (PI), which fluoresces red. Pretreatment of lymphocytes with CFDA did not affect their ability or interact with alloantibodies in the microcytotoxicity assays. When visually analyzed, detection of cell death by fluorescence was as sensitive as detection by eosin exclusion. However electronic detection of fluorescence was slightly more sensitive than visual detection. Automation of the fluorescent method required a calculation that converts electronic data to an ASHI score for cell death. One such method is described and evaluated. Both the automated and the conventional methods of analysis were used to obtain PRA values for various sera. There was good correlation between the PRA values obtained with the automated method versus the conventional method. Further, there was good correlation for PRA-derived alloantibody specificities obtained with the automated method versus the conventional method. These data demonstrate that automated fluorescence-based PRA analysis is an effective and practical alternative to conventional PRA analysis.

Microelectrophoresis for the separation of DNA fragments.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.

Cytomegalovirus detection in bone marrow transplant patients with idiopathic pneumonitis. A clinicopathologic study of the clinical utility of the polymerase chain reaction on open lung biopsy specimen tissue.

The morbidity and mortality rates of cytomegalovirus (CMV) infections, including pneumonitis in bone marrow transplant patients, are well documented and yet no rapid, sensitive diagnostic tool is available. To test the polymerase chain reaction as such a diagnostic tool, formalin-fixed, paraffin-embedded open lung biopsy material was selected from post-bone marrow transplant patients in whom pulmonary parenchymal changes were evident but viral inclusions were not seen. As a control, other immunosuppressed (non-bone marrow transplant) patients and low-risk nonimmunosuppressed patients were studied in a similar manner. Viral culture results and clinical data were available on all the high-risk patients. Two of 15 high-risk patients were found to have amplifiable CMV DNA despite the lack of histologic viral inclusions. One of these patients had been treated recently for CMV pneumonia, and the other had a positive culture of the lung specimens for CMV 13 days after biopsy. None of 12 control patients had amplifiable CMV DNA. These data indicate that the polymerase chain reaction is more sensitive than histologic examination and at least as sensitive as viral culture without evidence of false-positive results.

An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication.

A method was developed for fast and efficient isolation of DNA from formalin-fixed, paraffin-embedded tissue sections for subsequent use in PCRs and DNA hybridization assays. The method relies on the use of a sonicating water bath to disrupt tissue samples to which a small amount of micro-sized glass beads have been added. The sonicating glass beads provide fast and efficient physical shearing of fixed tissue sections, allowing for quick release and solubilization of the DNA. The extraction process from paraffin section to amplifiable target DNA takes 30 minutes. The method eliminates the need for repetitive solvent extractions and exhaustive proteinase K digestion. PCR amplification of human genomic and viral target sequences was successfully carried out on DNA isolated from a number of different types of normal and infected tissues.

Complementary carrier peptide synthesis: general strategy and implications for prebiotic origin of peptide synthesis.

A method for peptide synthesis is proposed based on a template-directed scheme that parallels that of the native ribosomal mechanism. In this procedure, peptide bond formation is facilitated by the juxtaposition of aminoacyl and peptidyl oligonucleotide carriers bound adjacent to one another on an oligonucleotide template. The general strategy of the synthesis and relevant model studies are described. The scheme provides an intrinsic mechanism by which oligonucleotides can direct the synthesis of polypeptides in the absence of protein or ribosomal machinery and, as such, suggests a model for the origin of prebiotic protein synthesis.