Printing of protein microarrays via a capillary-free fluid jetting mechanism.

Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer.

Picoliter-scale protein microarrays by laser direct write.

We demonstrate the accurate picoliter-scale dispensing of active proteins using a novel laser transfer technique. Droplets of protein solution are dispensed onto functionalized glass slides and into plastic microwells, activating as small as 50-microm diameter areas on these surfaces. Protein microarrays fabricated by laser transfer were assayed using standard fluorescent labeling techniques to demonstrate successful protein and antigen binding. These results indicate that laser transfer does not damage the active site of the dispensed protein and that this technique can be used to successfully fabricate a functioning protein microarray. Also, as a result of the efficient nature of the process, material usage is reduced by two to four orders of magnitude compared to conventional pin dispensing methods for protein spotting.

Splanchnic and mammary nitrogen metabolism by dairy cows fed dry-rolled or steam-flaked sorghum grain.

Objectives were to determine net release or uptake of alpha-amino N, ammonia N, and urea N across portal-drained viscera, liver, splanchnic, and mammary tissues of lactating Holstein cows (n = 8, 86 +/- 8 d in milk) fed alfalfa hay-based total mixed rations containing 40% dry-rolled or steam-flaked sorghum grain. The total mixed rations were offered at 12-h intervals in a crossover design. Blood samples were obtained from indwelling catheters in the portal, hepatic, and mammary veins and mesenteric or costoabdominal arteries, every 2 h for each cow and diet. Steam-flaking increased in vitro rate of starch hydrolysis compared with dry-rolled sorghum (66 vs. 25%). Diet did not alter dry matter intake (18.2 +/- 0.3 kg). Daily milk yield (27.6 +/- 0.8 kg), efficiency of production, and most milk components did not differ between diets, but fat yield was reduced (0.86 vs. 0.91 kg/d) by steam-flaked sorghum, and lactose concentration was increased (4.99 vs. 4.82%). Blood flows in portal and hepatic veins did not differ between diets. Steam-flaking tended to increase urea N cycling to the gut (162 vs. 95 g/d) compared with dry-rolling of sorghum, whereas net absorption of ammonia N and alpha-amino N across portal-drained viscera were decreased. Net mammary uptake of a-amino N increased more than 20% (83 vs. 67 g/d), resulting in a higher mammary extraction ratio (15 vs. 11%) for steam-flaked versus dry-rolled sorghum. Flaking of sorghum improved the efficiency of postabsorptive N metabolism by increasing urea N cycled to the gut and alpha-amino N uptake by the mammary gland.

Splanchnic and mammary nitrogen metabolism by dairy cows fed steam-rolled or steam-flaked corn.

Objectives were to determine net release or uptake of a-amino N, ammonia N, and urea N across portal-drained viscera, liver, splanchnic, and mammary tissues of lactating Holstein cows (n = 6; 109 +/- 9 d in milk) fed alfalfa hay-based total mixed rations (TMR) containing 40% steam-rolled or steam-flaked corn grain. The TMR were offered at 12-h intervals in a crossover design. Blood samples were obtained from indwelling catheters in portal, hepatic, and mammary veins and mesenteric or costo abdominal arteries, every 2 h for each cow and diet. Steam-flaked compared with steam-rolled corn greatly increased in vitro starch hydrolysis (56 vs. 34%). Daily intake of dry matter (18.4 +/- 0.4 kg/d), starch, N, and net energy for lactation by cows were not altered by processing corn; neither were daily yield of milk (29.1 +/- 0.7 kg/d), fat-corrected milk, nor fat-corrected milk per dry matter intake. Steam-flaking tended to increase percent milk protein (2.97 vs. 2.82%; P = 0.07), but not yield, and decrease percent lactose (4.83 vs. 4.94) but not yield. Portal and hepatic blood flows were not affected by diet, nor were net absorption of alpha-amino N and ammonia N. Steam-flaking compared with steam-rolling increased urea N cycling to portal-drained viscera (212 vs. 87 g/d) by 140%, estimated mammary uptake and extraction ratio of alpha-amino N. Flaking versus rolling of corn improved N utilization in dairy cows by increasing urea cycling to the gut and uptake of a-amino N by the mammary gland. Higher mammary uptake of alpha-amino N (78 vs. 50 g/d) by dairy cows fed steam-flaked corn tended to increase milk protein content and may explain the previously observed effects of cows fed steam-flaked versus steam-rolled corn.

Generation of mesoscopic patterns of viable Escherichia coli by ambient laser transfer.

We have generated mesoscopic patterns of viable Escherichia coli on Si(1 1 1), glass, and nutrient agar plates by using a novel laser-based transfer process termed matrix assisted pulsed laser evaporation direct write (MAPLE DW). We observe no alterations to the E. coli induced by the laser-material interaction or the shear forces during the transfer. Transferred E. coli patterns were observed by optical and electron microscopes, and cell viability was shown through green fluorescent protein (GFP) expression and cell culturing experiments. The transfer mechanism for our approach appears remarkably gentle and suggests that active biomaterials such as proteins, DNA and antibodies could be serially deposited adjacent to viable cells. Furthermore, this technique is a direct write technology and therefore does not involve the use of masks, etching, or other lithographic tools.