Method for printing functional protein microarrays

Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.     

SwizzleStick: A novel positive displacement microliter diluting device

The SwizzleStick, a simple positive displacement device for dispensing a microliter volume of sample from a microhematocrit capillary tube, is described. Because it is essentially a diluting device, a dye dilution test using both aqueous and protein-containing solutions of sodium sulfobromophthalein was devised to test the accuracy and precision of the SwizzleStick. The mean of the absorbances of 10 replicate 1 in 201 SwizzleStick dilutions, made by dispensing 20 μl of aqueous dye solution into 4.0 ml of buffer dispensed with a certified grade A volumetric pipet, was 0.216, SD 0.0005, CV 0.24% while the mean of the absorbances of 10 readings of a single bulk 1 in 201 dilution made with certified grade A glassware (1.0-ml volumetric pipet, 200-ml volumetric flask) was 0.216, SD 0.0004, CV 0.19%. Repeat studies using dye solution containing 6% human albumin showed that the volume dispensed by the SwizzleStick is not affected by the viscosity of plasma.     

Dispensing an enzyme-conjugated solution into an ELISA plate by adapting ink-jet printers

The rapid and precise delivery of small volumes of bio-fluids (from picoliters to nanoliters) is a key feature of modern bioanalytical assays. Commercial ink-jet printers are low-cost systems which enable the dispensing of tiny droplets at a rate which may exceed 10⁴ Hz per nozzle. Currently, the main ejection technologies are piezoelectric and bubble-jet. We adapted two commercial printers, respectively a piezoelectric and a bubble-jet one, for the deposition of immunoglobulins into an ELISA plate. The objective was to perform a comparative evaluation of the two classes of ink-jet technologies in terms of required hardware modifications and possible damage on the dispensed molecules. The hardware of the two printers was modified to dispense an enzyme conjugate solution, containing polyclonal rabbit anti-human IgG labelled with HRP in 7 wells of an ELISA plate. Moreover, the ELISA assay was used to assess the functional activity of the biomolecules after ejection. ELISA is a common and well-assessed technique to detect the presence of particular antigens or antibodies in a sample. We employed an ELISA diagnostic kit for the qualitative screening of anti-ENA antibodies to verify the ability of the dispensed immunoglobulins to bind the primary antibodies in the wells. Experimental tests showed that the dispensing of immunoglobulins using the piezoelectric printer does not cause any detectable difference on the outcome of the ELISA test if compared to manual dispensing using micropipettes. On the contrary, the thermal printhead was not able to reliably dispense the bio-fluid, which may mean that a surfactant is required to modify the wetting properties of the liquid.     

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section¹. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).     

Modification of an automated liquid-handling system for reagent-jet, nanoliter-level dispensing

Reducing the scale of biochemical reactions is becoming commonplace. Examples include the screening of large libraries of chemical compounds or gene sequences. These applications demand the ability to transfer sub-microliter volumes of fluid. To this end, we have modified a Hamilton MICROLAB 2200 with high-speed solenoids and a liquid pressurization system to modulate volume delivery down to the nanoliter level. Additional modifications include the use of sapphire-tipped dispensing nozzles and a high-resolution substage to assist in the construction of DNA microarrays. Techniques for characterizing the dispensed volume are presented.     

Enzyme microarrays assembled by acoustic dispensing technology

Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Through the use of acoustic dispensing technology, nanodroplets containing 10 μM ATP (3 μCi/μL ³²P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40-nL compartments (100 droplets/slide) for Pim1 (proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 μM substrate. The microarray was incubated at 30 °C (97% R_h) for 1.5 h. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. With phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 to 3 μM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165, and average coefficients of variation for the assay were ∼20%. Coefficients of variation for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development.     

Downsizing proteolytic digestion and analysis using dispenser-aided sample handling and nanovial matrix-assisted laser/desorption ionization-target arrays

An efficient technique for enzymatic digestion of proteins in nanovial arrays and identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization (MALDI-MS) is presented in this work. Through dispensing of a protein solution with simultaneous evaporation the protein (substrate) is concentrated up to 300 times in-vial. At higher substrate concentrations the catalytic turnover numbers increase according to the Michaelis-Menten kinetics. Therefore, the dispenser-aided nanodigestion is valuable for identification of low-level proteins (10 nM-500 nM) as well as for automatic high efficiency digestions performed in 0.2-10 min. As an example of low-level protein identification, a 10 nM solution of lysozyme C was unambiguously identified after 5 min of nanodigestion. Moreover, only 30 s nanodigestion was sufficient to identify hemoglobin (10 microM), exemplifying the fast catalysis of the nanodigestion technique. The developed silicon flow-through piezoelectric dispenser is adapted for low-volume and preconcentrated samples in the nL-microL range and provides fast, accurate and contact-free sample positioning into the nanovials. In this work, the properties of the nanodigestion concept regarding proteins of different characteristics are explored. Furthermore, the potential of automated protein identification using precoated proteolytic nanovial-arrays is demonstrated.     

Recovery of copper and cobalt by biopolymer gels

The recovery of copper from synthetic aqueous media circulating in a loop fluidized bed reactor operated batchwise was investigated by using the following biopolymer systems: (1) a viscous solution of sodium alginate (from kelp) dispensed directly into the reactor fluid containing dissolved copper (sulfate salt) at initial concentrations of 60-200 ppm, (2) partially coagulated calcium alginate spheres for absorbing dissolved copper at initial concentrations of 10-40 ppm, and (3) a mixture of green algae Microcystis and sodium alginate dispensed directly into the reactor fluid. The recovery of copper and cobalt, a strategic metal, from cobalt ore leachate was achieved by a two-step approach: direct dispensing of sodium alginate to absorb the bulk of metals followed by the addition of partially coagulated calcium alginate spheres to "polish" the leachate. Metal binding capacity and conditional stability constant of each biopolymer system as well as the effective diffusivity of cupric ion in the matrix of biopolymer gels are reported.     

Reducing mortality from anthrax bioterrorism: strategies for stockpiling and dispensing medical and pharmaceutical supplies

A critical question in planning a response to bioterrorism is how antibiotics and medical supplies should be stockpiled and dispensed. The objective of this work was to evaluate the costs and benefits of alternative strategies for maintaining and dispensing local and regional inventories of antibiotics and medical supplies for responses to anthrax bioterrorism. We modeled the regional and local supply chain for antibiotics and medical supplies as well as local dispensing capacity. We found that mortality was highly dependent on the local dispensing capacity, the number of individuals requiring prophylaxis, adherence to prophylactic antibiotics, and delays in attack detection. For an attack exposing 250,000 people and requiring the prophylaxis of 5 million people, expected mortality fell from 243,000 to 145,000 as the dispensing capacity increased from 14,000 to 420,000 individuals per day. At low dispensing capacities (<14,000 individuals per day), nearly all exposed individuals died, regardless of the rate of adherence to prophylaxis, delays in attack detection, or availability of local inventories. No benefit was achieved by doubling local inventories at low dispensing capacities; however, at higher dispensing capacities, the cost-effectiveness of doubling local inventories fell from 100,000 US dollars to 20,000 US dollars/life year gained as the annual probability of an attack increased from 0.0002 to 0.001. We conclude that because of the reportedly rapid availability of regional inventories, the critical determinant of mortality following anthrax bioterrorism is local dispensing capacity. Bioterrorism preparedness efforts directed at improving local dispensing capacity are required before benefits can be reaped from enhancing local inventories.     

Capacity of aquatic bacteria to act as recipients of plasmid DNA.

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.     

Capacity of aquatic bacteria to act as recipients of plasmid DNA

A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer.     

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.     

A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.     

Theoretical analysis of thermal damage in biological tissues caused by laser irradiation

A bioheat transfer approach is proposed to study thermal damage in biological tissues caused by laser radiation. The laser light propagation in the tissue is first solved by using a robust seven-flux model in cylindrical coordinate system. The resulting spatial distribution of the absorbed laser energy is incorporated into the bioheat transfer equation for solving temperature response. Thermal damage to the tissue is assessed by the extent of denatured protein using a rate process equation. It is found that for the tissue studied, a significant protein denaturation process would take place when temperature exceeds about 53 degrees C. The effects of laser power, exposure time and beam size as well as the tissue absorption and scattering coefficients on the thermal damage process are examined and discussed. The laser conditions that cause irreversible damage to the tissue are also identified.     

Residual water modulates QA- -to-QB electron transfer in bacterial reaction centers embedded in trehalose amorphous matrices

The role of protein dynamics in the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), was studied at room temperature in isolated reaction centers (RC) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in trehalose water systems of different trehalose/water ratios. The effects of dehydration on the reaction kinetics were examined by analyzing charge recombination after different regimes of RC photoexcitation (single laser pulse, double flash, and continuous light) as well as by monitoring flash-induced electrochromic effects in the near infrared spectral region. Independent approaches show that dehydration of RC-containing matrices causes reversible, inhomogeneous inhibition of Q(A)(-)-to-Q(B) electron transfer, involving two subpopulations of RCs. In one of these populations (i.e., active), the electron transfer to Q(B) is slowed but still successfully competing with P(+)Q(A)(-) recombination, even in the driest samples; in the other (i.e., inactive), electron transfer to Q(B) after a laser pulse is hindered, inasmuch as only recombination of the P(+)Q(A)(-) state is observed. Small residual water variations ( approximately 7 wt %) modulate fully the relative fraction of the two populations, with the active one decreasing to zero in the driest samples. Analysis of charge recombination after continuous illumination indicates that, in the inactive subpopulation, the conformational changes that rate-limit electron transfer can be slowed by >4 orders of magnitude. The reported effects are consistent with conformational gating of the reaction and demonstrate that the conformational dynamics controlling electron transfer to Q(B) is strongly enslaved to the structure and dynamics of the surrounding medium. Comparing the effects of dehydration on P(+)Q(A)(-)-->PQ(A) recombination and Q(A)(-)Q(B)-->Q(A)Q(B)(-) electron transfer suggests that conformational changes gating the latter process are distinct from those stabilizing the primary charge-separated state.     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.