Whole-cell biochips for bio-sensing: integration of live cells and inanimate surfaces

Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.     

Whole-cell biochips for online water monitoring

Chip-integrated luminescent recombinant reporter bacteria were combined with fluidics and light detection systems to form a real-time water biomonitor. The biomonitor was exposed to a continuous water flow for up to ten days, in the course of which it was challenged with spikes of both model toxic compounds and toxic environmental samples. All simulated contamination events were reported within 0.5-2.5?h. Furthermore, the response pattern of the reporter bacteria was indicative of the nature of the contaminating chemicals. Efforts were aimed at improving signal quality and at the development of an alarm management software. Following further research, a device of the proposed design could be implemented in monitoring networks as an early warning system against water pollution by toxic chemicals.     

Three-dimensional focusing of red blood cells in microchannel flows for bio-sensing applications

Three-dimensional (3D) focusing of particles in microchannels has been a long-standing issue in the design of biochemical/biomedical microdevices. Current microdevices for 3D cell or bioparticle focusing involve complex channel geometries in view of their fabrication because they require multiple layers and/or sheath flows. This paper proposes a simple method for 3D focusing of red blood cells (RBCs) in a single circular microcapillary, without any sheath flows, which is inspired from the fluid dynamics phenomenon in that a spherical particle lagging behind a Poiseuille flow migrates toward and along the channel axis. More explicitly, electrophoresis of RBCs superimposed on the pressure-driven flow is utilized to generate an RBC migration mode analogous to this phenomenon. A particle-tracking scheme with a sub-pixel resolution is implemented to spatially position red blood cells flowing through the channel, so that a probability density function (PDF) is constructed to evaluate the tightness of the cell focusing. Above a specific strength of the electric field, approximately 90% of the sheep RBCs laden in the flow are tightly focused within a beam diameter that is three times the cell dimension. Particle shape effect on the focusing is discussed by making comparisons between the RBCs and the spherical particles. The lateral migration velocity, predicted by an existing theoretical model, is in good agreement with the present experimental data. It is noteworthy that 3D focusing of non-spherical particles, such as RBCs, has been achieved in a circular microchannel, which is a significant improvement over previous focusing methodologies.     

Immunological biochips for parallel detection of surface antigens and morphological analysis of cells

A biochip for detecting 26 cluster differentiation (CD), HLA-DR and IgM antigens on lymphocyte surface is described. The biochip, which represents a microarray of antibodies (IgG) against a panel of selected antigens immobilized on transparent plastic surfaces in 1.5-mm spots, was used for the study of normal and neoplastic lymphocytes and can also be used for determining percent of cells expressing definite surface antigens in lymphocyte suspensions. The results are consistent with data obtained by flow cytometry. The novel biochip technology entails a combination of conventional staining of cells immobilized on biochips and morphological analysis.     

Detection of KRAS mutations in tumor cells using biochips

Somatic mutations in the KRAS gene are important markers of some types of tumors, for example, pancreatic cancer, and may be useful in early diagnostics. A biochip has been developed which allows determining most frequent mutations in 12, 13 and 61 codons of the KRAS gene. To increase the sensitivity of the method and to make possible the analysis of minor fractions of tumor cells in clinical samples the method of blocking a wild type sequence PCR amplification by LNA-oligonucleotides has been used. The product of LNA-clamp PCR was further hybridized with oligonucleotide probes, immobilized on biochip. Biochip was tested with 42 clinical DNA samples from patients with pancreatic cancer, mostly ductal adenocarcinomas. As reference methods, the RFLP analysis and sequencing were used. The developed approach allows detecting somatic mutations in the KRAS gene if the portion of tumor cells with mutation is at least 1% of whole cell population.     

Survival of human rhinovirus type 14 dried onto nonporous inanimate surfaces: effect of relative humidity and suspending medium

To study the survival of human rhinovirus 14 on environmental surfaces, each stainless steel disk (1 cm in diameter) was contaminated with 10 microL (about 10(5) plaque-forming units) of the virus suspended in either 1 chi tryptose phosphate broth (TPB), 5 mg/mL of bovine mucin in normal saline, or undiluted human nasal discharge. The inoculum was dried in a laminar flow cabinet for 1 h under ambient conditions. The disks were then placed in a glass chamber (20 +/- 1 degree C) with the relative humidity at either low (20 +/- 5%), medium (50 +/- 5%), or high (80 +/- 5%) level. At appropriate intervals, the disk to be tested was placed in 1 mL of tryptose phosphate broth and the eluate titrated in A-5 HeLa cells. When the virus was suspended in either tryptose phosphate broth, mucin, or the nasal discharge and subjected to initial drying, there was a 3.0 +/- 1.0, 82.0 +/- 6.7, and 89.0 +/- 3.0% loss in virus infectivity, respectively. The half-life of the TPB-suspended virus was about 14 h at the high relative humidity as compared with less than 2 h at the other two relative humidity levels. The half-lives for the mucin-suspended virus at the high, medium, and low relative humidity were 1.42, 0.55, and 0.24 h, respectively; the corresponding values for the nasal discharge suspended virus being 0.17, 0.25, and 0.09 h.     

Genetic tags for labelling live cells: gap junctions and beyond

The availability of green fluorescent protein (GFP) as a tracer for observing proteins in living cells has revolutionized cell biology and spurred an intensive search for GFP variants with novel characteristics, additional autofluorescent proteins and alternative techniques of protein labelling. Two recent studies - one on tagging with tetracysteine motifs and labelling with biarsenic fluorophores of different colours, and the other on GFP tagging and fluorescence recovery after photobleaching (FRAP) - show how membrane channels are added and removed from gap junctions by using different fluorescent tags to distinguish between newly synthesized and older protein populations.     

Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells

Summary Whole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture. In these protoplasts the membrane resting potential (V _m ) was found to be –59±23 mV (n=23) in 1mm K _o ^– . The meanV _m became more negative as [K^–] _o decreased, but was more positive than the K⁺ equilibrium potential. There was no evidence of electrogenic pump activity. We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltagegated channel activity. Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K⁺-selective channels. A local minimum in the outward current-voltage curve nearV _m =150 mV suggests that these currents are mediated by two populations of K⁺-selective channels. The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca²⁺ into the cytoplasm. We identify unitary currents from two K⁺-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current. Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current. Current activation is fast and follows an exponential time course. The current saturates and in some cases decreases at membrane potentials more negative than –175 mV. This current is conducted by poorly selective K⁺ channels, whereP _Cl/P _K=0.43±0.15. We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized. It is possible that these channels mediate inward whole-cell current. When the membrane is hyperpolarized to potentials more negative than –250 mV large, irregular inward current is activated. A third type of inward whole-cell current is briefly described. This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials –90<V _m <0 mV.     

Molecular recognition of cocaine by acetylcholinesterases for affinity purification and bio-sensing

Cholinesterases can be used as sensitive biorecognition elements for widely used agricultural pesticides. This requires highly purified and inhibitor-free enzyme preparations. In the present work the cocaine derivative benzoylecgonine was for the first time used as the molecular recognition element for the purification of acetylcholinesterase from Electrophorus electricus by affinity chromatography. The preparation of enriched enzyme without the contamination by an inhibitor, which is traditionally used for eluting the "affinity" bound protein, was achieved. The specific activity was 2.2-fold increased to 3100 Umg(-1). The same cocaine derivative was immobilized on the surface of a piezoelectric crystal in order to analyze the binding of acetylcholinesterases from two different species, E. electricus and Drosophila melanogaster, to the immobilized inhibitor. Evaluation of the binding curves allowed the analysis of the binding kinetics. These experiments are fundamental for the development of a (competitive) biosensor for inhibitors of cholinesterase.     

Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes.

Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization with the fluorescently labelled probes required several modifications of standard procedures. Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol. Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.85% NaCl. Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound. The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash the labelled probes out of the cells. Stable labelling could be obtained with rhodamine-labelled probes when the specimen was mounted in immersion oil, and high hybridization intensities of the Methanosarcina cells were found even in the presence of biomass from an anaerobic reactor. The inherent high autofluorescence of the biomass could be lowered by use of a highly specific narrow-band filter. The probes were found to be specific for Methanosarcina and useful for detection of this genus in samples from anaerobic reactors.     

Immunological biochips for studies of human erythrocytes

Immunological microarrays (biochips) for detecting erythrocyte surface antigens, viz., blood group antigens (A, B, 0) and Rhesus system antigens (D, E, e, C, and c), are described. The biochips represent transparent plastic supports onto which 1.5-mm spots of specific immobilized antibodies (IgM) are coated in different dilutions. The volume of tested blood samples is rather small (1–2 μl). Binding of erythrocytes to antibodies immobilized on the biochips is specific and allows further morphological analysis of bound cells. Analysis of the dynamics of cell detachment from biochip spots using a microfluidic chamber at different flow rates of the washing solution showed that combination of a biochip with a microfluidic chamber is a promising approach to concentration of cells of various immunotypes even if their content in the mixture is very low.     

Whole-cell voltage clamp and intracellular perfusion technique on single smooth muscle cells

Using freshly isolated single smooth muscle cells prepared by collagenase treatment, membrane currents were recorded by whole-cell voltage clamp. Intracellular constituents were modified by using an intracellular perfusion technique, i.e., pipette solutions were continuously exchanged from control to test solutions during current recording. In smooth muscle cells, intracellular application of ATP, but not cyclic AMP, enhanced the amplitude of Ca2+ currents and prevented current run-down. In addition, with this stabilization of Ca2+ current recording by ATP, introduction of various chemicals into the cell using the intracellular perfusion technique is useful for investigations of regulation of ion channels in smooth muscle cells.     

Whole-cell voltage clamp and intracellular perfusion technique on single smooth muscle cells

Using freshly isolated single smooth muscle cells prepared by collegenase treatment, membrane currents were recorded by whole-cell voltage clamp. Intracellular constituents were modified by using an intracellular perfusion technique, i.e., pipette solutions were continuously exchanged from control to test solutions during current recording. In smooth muscle cells, intracellular application of ATP, but not cyclic AMP, enchanced the amplitude of Ca²⁺ currents and prevented current run-down. In addition, with this stabilization of Ca²⁺ current recording by ATP, introduction of various chemicals into the cell using the intracellular perfusion technique is useful for investigations of regulation of ion channels in smooth muscle cells.     

The role of integration and clonal expansion in HIV infection: live long and prosper

Integration of viral DNA into the host genome is a central event in the replication cycle and the pathogenesis of retroviruses, including HIV. Although most cells infected with HIV are rapidly eliminated in vivo, HIV also infects long-lived cells that persist during combination antiretroviral therapy (cART). Cells with replication competent HIV proviruses form a reservoir that persists despite cART and such reservoirs are at the center of efforts to eradicate or control infection without cART. The mechanisms of persistence of these chronically infected long-lived cells is uncertain, but recent research has demonstrated that the presence of the HIV provirus has enduring effects on infected cells. Cells with integrated proviruses may persist for many years, undergo clonal expansion, and produce replication competent HIV. Even proviruses with defective genomes can produce HIV RNA and may contribute to ongoing HIV pathogenesis. New analyses of HIV infected cells suggest that over time on cART, there is a shift in the composition of the population of HIV infected cells, with the infected cells that persist over prolonged periods having proviruses integrated in genes associated with regulation of cell growth. In several cases, strong evidence indicates the presence of the provirus in specific genes may determine persistence, proliferation, or both. These data have raised the intriguing possibility that after cART is introduced, a selection process enriches for cells with proviruses integrated in genes associated with cell growth regulation. The dynamic nature of populations of cells infected with HIV during cART is not well understood, but is likely to have a profound influence on the composition of the HIV reservoir with critical consequences for HIV eradication and control strategies. As such, integration studies will shed light on understanding viral persistence and inform eradication and control strategies. Here we review the process of HIV integration, the role that integration plays in persistence, clonal expansion of the HIV reservoir, and highlight current challenges and outstanding questions for future research.     

Lysozyme for capture of microorganisms on protein biochips

Lysozyme placed on the SiO₂ surfaces that have previously been derivatized with C₁₈ coating will capture both Escherichia coli and Listeria monocytogenes cells from PBS buffer at pH 7.2. This phenomenon is of significance for the design and fabrication of protein biochips that are designed to capture bacteria from buffer or water so that these can be further interrogated with respect to possible pathogenicity. Fluorescent microscopy shows that two types of bacteria (gram-negative E. coli and gram-positive Listeria spp.) will be adsorbed by lysozyme placed on the surface of the biochip but that strong adsorption of the bacteria is reduced but not eliminated when Tween 20 is present (at 0.5%) in the PBS buffer in which the cells are suspended. In comparison, Tween 20 and Bovine Serum Albumin (BSA) almost completely block adsorption of these bacteria on C₁₈ coated surfaces. The combination of a lysozyme surface with Tween 20 gives a greater degree of adsorption of L. monocytogenes than E. coli, and hence suggests selectivity for the more hydrophobic E. coli may be reduced by the Tween 20. This paper presents protocols for preparing protein-coated, SiO₂ surfaces and the effect of buffer containing Tween 20 on adsorption of bacteria by SiO₂ surfaces coated with C₁₈ to which BSA, lysozyme or C11E9 antibody is immobilized at pH 7.2 and ambient temperature.     

Barotaxis: How cells live and move under pressure

Cell migration is an essential process that controls many physiological functions ranging from development to immunity. In vivo, cells are guided by a combination of physical and chemical cues. Chemokines have been the center of attention for years, but the role of physical properties of tissues has been under-investigated, despite the fact that these properties can be drastically modified in pathology. Here, we discuss the role of one important tissue physical property, hydraulic resistance, in cell guidance, a phenomenon referred to as barotaxis, and describe the underlying physical principles and molecular mechanisms. Finally, we speculate on the putative role of barotaxis in physiological processes involving immune and cancer cells.     

Whole-cell currents in single and confluent M-1 mouse cortical collecting duct cells

M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl- Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M- 1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.