Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Automatic analytical monitor and control of industrial process
Reexamination Certificate
2000-03-13
2002-09-10
Drodge, Joseph W. (Department: 1723)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Automatic analytical monitor and control of industrial process
C422S063000, C422S082050, C422S105000, C422S105000, C436S043000, C436S180000, C382S129000
Reexamination Certificate
active
06447723
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to microarray spotting instruments. More particularly, the present invention relates to improved spotting instruments that incorporate sensors and methods of using those sensors for improving performance of the spotting instruments.
As is well known (and described for example in U.S. Pat. No. 5,807,522 to Brown et al. and in “DNA Microarrays: A Practical Approach”, Schena, Mark, New York, Oxford University Press, 1999, ISBN 0-19-963776-8), microarrays are arrays of very small samples of purified DNA or protein target material arranged as a grid of hundreds or thousands of small spots on a solid substrate. When the microarray is exposed to selected probe material, the probe material selectively binds to the target spots only where complementary bonding sites occur, through a process called hybridization. Subsequent quantitative scanning by a fluorescent microarray scanner (i.e., a scanning instrument) may be used to produce a pixel map of fluorescent intensities (See, e.g., U.S. Pat. No. 5,895,915, to DeWeerd et al.). This fluorescent intensity map can then be analyzed by special purpose quantitation algorithms which reveal the relative concentrations of the fluorescent probes and hence the level of gene expression, protein concentration, etc., present in the cells from which the probe samples were extracted.
The microarray substrate is generally made of glass which has been treated chemically to provide for molecular attachment of the spot samples of microarray target material. The microarray substrate is also generally of the same size and shape as a standard microscope slide, about 25 mm×75 mm×1 mm thick. The array area can extend to within about 1.5 mm of the edges of the substrate, or can be smaller. The spots of target material (typically DNA) are approximately round. The spot diameter is generally determined by the dispensing or spotting technique used and typically varies from about 75 microns to about 500 microns, and may be as small as about 20 microns. The general trend is toward smaller spots, which produce more compact arrays. The center-to-center spacing between the spots usually falls into the range of 1.5 to 2.5 spot diameters.
FIG. 1A
, which is not drawn to scale, shows a top view of a prior art microarray
100
. In
FIG. 1A
, each of the circles represents a tiny spot of target material that has been deposited onto a rectangular glass substrate
101
, and the spots are shown in a magnified view as compared to the substrate
101
. Assuming typical dimensions of 100 &mgr;m spot diameter and 200 &mgr;m center-to-center spacing between the spots, the illustrated six by six array of spots covers only a 1100 &mgr;m by 1100 &mgr;m square area of the 25 mm by 75 mm area defined by the substrate
101
. Thousands of spots are usually deposited in a typical microarray and the spots may cover nearly the entire substrate. The portion of the microarray that is covered with spots of target material may be referred to as the “active area” of the microarray.
There are several well known methods of depositing the spots onto the substrate of a microarray, and instruments that deposit the spots are typically referred to as “spotting instruments”. One popular method is to use one or more “pins” to transfer the target material from a reservoir onto the microarray substrate.
FIG. 1B
shows an example of such a prior art pin
102
, which includes a pin head
104
and a shaft
106
. Both the pin head
104
and the shaft
106
are generally cylindrical, and the pin head
104
and shaft
106
are generally disposed so that they are coaxial. The diameter of the pin head
104
is greater than the diameter of the shaft
106
, and the shaft is substantially longer than the pin. One end
107
of the shaft
106
is tapered or sharpened, and the other end of the shaft is attached or bonded to the pin head
104
. Examples of such pins are described in, for example, U.S. Pat. No. 5,770,151 (Roach et al.) and U.S. Pat No. 5,807,522 (Brown et al.).
In operation, the sharp ends
107
of the pins are dipped into a reservoir of the liquid target material so that some of the material is “collected by” or becomes attached to the pins. The sharp ends of the pins are then placed in contact with the substrate to deposit tiny amounts of the material onto selected locations of the substrate. The pins are normally moved by a mechanical or robotic apparatus so the spots may be accurately placed at desired locations on the substrate.
Some types of pins are capable of collecting only enough target material to form a single spot on the microarray before they need to be re-dipped in the reservoir, whereas others can collect enough target material from the reservoir to form several or even hundreds of spots before they need to be re-dipped in the reservoir. In either case, the pins must be manufactured to very precise tolerances to insure that each spot formed by the pin will be of controlled size. As a result of these demanding specifications, the pins are rather expensive (e.g., a single pin typically costs several hundred dollars). Also, the sharp ends of the pins are so small and precisely shaped (e.g., a square tip measuring 50 microns on a side) that the pins are fragile. Accordingly, to prevent damage, the sharp ends of the pins should only be subjected to a tiny force when the sharp ends are placed in contact with the substrate or any other solid object.
Spotting instruments typically form microarrays in batches. For example, in a single “run”, a spotting instrument may form up to one hundred identical microarrays. After forming enough spots of a particular target material to complete the batch of microarrays being spotted, the pins generally need to be washed (to remove any excess liquid target material), and then dried before they can be dipped into another reservoir of target material. So the process of forming microarrays with a “pin-type” spotting instrument includes steps of (1) positioning a pin over a reservoir of target material; (2) dipping the sharp end of the pin into the reservoir; (3) withdrawing the sharp end of the pin from the reservoir; (4) moving the pin over a selected location within the active area of a microarray; (5) lowering the pin to bring the sharp end of the pin into contact with the microarray substrate to form a single spot of controlled size at the selected location; (6) raising the pin to separate the sharp end of the pin from the substrate; (7) repeating steps (4), (5), and (6) until the pin's supply of target material is exhausted or until the desired number of spots have been placed on the bach of microarrays being produced; (8) washing the pin by either placing the pin in a stream of cleaning solution or by dipping the pin into a reservoir of cleaning solution; and (9) drying the pin. The spotting instrument repeats all of these steps numerous times to form a single microarray.
Since microarrays typically include thousands of spots, using only a single pin to form the microarray would be extremely time consuming. Accordingly, spotting instruments are often capable of simultaneously manipulating several pins.
FIGS. 1C
,
1
D, and
1
E show side, top, and perspective, views respectively of a printhead
110
that can simultaneously hold sixteen pins
102
. Printhead
110
is a solid block of material, typically metal, that defines an array of sixteen apertures
112
. The apertures
112
are slightly larger than the outer diameter of the shafts
106
so the shafts can extend through the apertures
112
. The apertures
112
are also smaller than the outer diameter of the pin heads
104
so that when the shaft of a pin is dropped into one of the apertures
112
, the pin head
104
will be supported by the upper surface of the printhead
110
. The pins are thereby “slip-fit” into the apertures of the printhead.
FIGS. 1F and 1G
show side and top views, respectively, of sixteen pins mounted into printhead
110
.
FIG. 1H
illustrates printhead
110
being lowered to place the sharp ends of the pins
Phaff Mona L.
Schermer Mack J.
Drodge Joseph W.
Hale & Dorr LLP
Packard Instrument Company Inc.
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