Microarray technology is the most successful example of miniaturization in modern life science. Taking advantage of high parallelism resulting from miniaturization microarrays has become a tool of choice in numerous applications (1). However in contrast to microarrays themselves, the methods of their manufacturing are generally completely macroscopic.

Currently, most microarrays are produced by pin or piezo-jet technology. Both use 384-well (seldom 96-well) microtiter plates filled with the solutions to be spotted. Pins or piezo dispensers are placed in the holder so that they match the pattern of the plate (i.e., in the case of a 384-well plate, they have a 4.5-mm pitch). This imposes some restrictions on the shape, size, and density of arrays produced in multipin mode. These arrays consist of clusters of spots matching the pattern of the microtiter plate they were printed from. For example, no microarray smaller than 4.5 × 4.5 mm can be printed in multipin mode. On the other hand, in a highly parallel mode with 48 (typically 4 × 12) pins, the resultant array will cover the entire 25 × 75 mm microscopic slide, which is unacceptable for 100-element arrays (i.e., these arrays are currently produced in low-throughput single pin mode).

A few exceptions from this general trend are based on parallel depositing of presynthesized species by contact (2-5) or noncontact (6-9) method. Both exploit planar microcapillary chips with an array of spotting or shooting nozzles. Although these methods provide a high-throughput for low-complexity arrays, they require special machinery and are not easily compatible with existing microarray spotters available in many biological laboratories.

Here we propose a spotter-compatible microstamping tool capable of printing up to 127 spots within a 3 × 3 mm are in single touch. We use it for production of a 70-element hydrogel-based microarray for identification of drug-resistant strains of Mycobacterium tuberculosis (10).

The key element of our printing tool is a glass multicapillary funnel with 127 microchannels forming a hexagonal array (Figure 1A). The funnel was manufactured at an industrial environment [Technology and Equipment for Glass Structures (TEGS), Saratov, Russian Federation] by forming a hexagonal array of glass tubes and baking them together. Then this multichannel structure was drawn in a special oven to obtain the funnel of desired shape. This technology was used earlier, for example, for the manufacturing of polycapillary lenses (11).

Figure 1. Multicapillary printing tool and spotted arrays. (Click to enlarge)

At the broad end of the funnel, the diameter of the microchannels is 1 mm with a 1.3-mm pitch. At the narrow end of the funnel, the diameter is 0.21 mm with a 0.27-mm pitch, and the funnel's length is 64.5 mm. These microchannels serve as guides for polypropylene capillaries used for printing, (Figure 1, B and C). They were drawn manually from 1-mL insulin syringes (Becton Dickinson, Franklin Lakes, NJ, USA). Every capillary was inserted into the funnel and adjusted so that its outer diameter at the narrow end of funnel (monitored by means of optical microscope) was from 0.15 to 0.18 mm and then trimmed at this position by a sharp blade (Stanley Works, Sheffield, England). The blade sharpness is essential, as it is a major factor affecting the quality of the printing end of the capillary, so the blades should be replaced as soon as necessary. At the opposite end, every capillary was trimmed to achieve the necessary length (from 65 to 66 mm, depending on its position in the funnel). Then the capillaries were filled with spotting solutions and inserted into the microchannels again. At the broad end of funnel, the capillaries were blocked from moving out by an upper clamp (Figure 1A), so that they protrude from the narrow end of funnel by approximately 0.5 mm. The upper clamp is normally tightly attached to the funnel and removed only to access the capillaries. This makes the capillary working like a spring: when it touches the surface of substrate with its protruding end, it shrinks, exerting some pressure and forming the tight contact necessary to transfer liquid to the surface.

The entire assembly was placed into a QArray spotter (Genetix, Hampshire, England) instead of a standard pin tool by means of a specially designed holder (see Supplementary Figure S1 available online at www.BioTechniques.com). In the course of printing, this stamp was brought in contact with slides, so that it stamped the pattern formed by the array of microcapillaries (Figure 1D).