Biochip reader with enhanced illumination and bioarray positioning apparatus

The United States Government has rights in this invention pursuant to Contract No. W-31-109-ENG-38 between the United States Government and Argonne National Laboratory.

The present invention relates to a biochip reader having a novel method of illumination and improved illumination and bioarray positioning apparatus for enhanced quantitative analysis of biochip data.

Analysis of biochip or bioarray data is carried out by the detection of the fluorescence from labeled target molecules that specifically interact with an immobilized array of molecular probes. The molecular probes may be attached directly onto a glass substrate or the probes may be attached onto a transparent plastic substrate. In an Argonne National Laboratory (ANL) 3D bioarray, the arrayed probes are attached to the glass substrate through a porous carrier which is chemically bound to the glass substrate.

One of the major problems in the quantitative analysis of bioarray data is finding a method of illumination of the array that is uniform over the area of the array to be analyzed. Any non-uniformity in the illumination translates into differences in the intensity of the fluorescence and thus tends to lead to erroneous results.

Another problem is positioning a glass substrate within an optical pathway so that the bioarray will be in the focal plane of a lens/lens array and within the field of view of the reader's optical system. Positioning the glass substrate has to be done repeatedly and with ease, without causing damage to the bioarray or the glass substrate. Positioning the glass substrate should not depend on the regular microscopic glass substrate thickness variation, typically, for example, 0.97 mm to 1.1 mm and also, length and width variations, length typically, for example, 75.513 mm to 76.2 mm, and width typically, for example, 24.638 mm to 25.552 mm. Information about commercially available microscope slides can be obtained, for example, at http://www.corning.com; and http://www.tedpella.com/histo_html/slides.htm. Positioning the glass substrate should not depend on whether or not a bioarray is covered with a reaction chamber. Reaction chamber information can be obtained, for example, at http://www.gracebio.com, http://www.eppendorf.com; http://www.mjr.com; and http://www.fishersci.com.

A need exists for an improved method of illumination and illumination apparatus to enable enhanced quantitative analysis of biochip data. It is desirable to provide such method of illumination and illumination apparatus that is effective and that is generally inexpensive, portable, lightweight, and simple to implement.

A need exists for an improved mechanism for positioning a glass substrate within an optical pathway so that the bioarray will be in the focal plane and within the field of view of an optical system. It is desirable to provide such an improved mechanism that is easy to use without causing any damage to the bioarray or the glass substrate.

A principal object of the present invention is to provide a biochip reader having an enhanced method of illumination and improved illumination and bioarray positioning apparatus enabling enhanced quantitative analysis of bioarray data. Other important objects of the present invention are to provide a method and apparatus for illumination in a biochip reader substantially without negative effect; and that overcome some disadvantages of prior art arrangements.

In brief, a novel method of illumination and illumination and bioarray positioning apparatus are provided in a biochip reader. Illumination is provided, for example, by a non-collimated laser source or a diode source. The light is directed to opposing sides of a glass substrate by a pair of optical fiber bundles. The glass substrate carries a bioarray. Each of the optical fiber bundles are splayed out to make a fan, the fan being one fiber thick and defining a line of optical fiber faces. This process randomizes any non-uniformity in the illumination source, creating a more uniform illumination of the bioarray. A respective divergent diffuser is provided proximate to each row of optical fiber faces coupling and diffusing light substantially evenly through the opposing sides of the glass substrate to illuminate the bioarray supported by the glass substrate.

The biochip reader includes illumination apparatus, a glass holder, and an optical system. The glass holder supports and aligns the glass substrate carrying the bioarray with the optical system. The glass holder includes a plastic springs member in contact engagement with the glass substrate with low contact forces for positioning the bioarray in a focal plane of the optical system.

In accordance with features of the invention, a manual positioner is coupled to the glass holder for simply positioning the bioarray within the field of view of an optical system. The divergent diffusers separate the optical fiber faces from the edges of the glass substrate, protecting the optical fibers from mechanical damage. A second function of the divergent diffusers is to reflect back outwardly going light to the glass to increase illumination efficiency. The glass substrate functions as a secondary light guide. The optical fiber bundles directing the laser light to the glass substrate are, for example, borosilicate fiber light guides. The optical fiber bundles also can be, for example, quartz, or plastic fiber light guides. Light also can be directed to opposing ends of the glass substrate by a second pair of optical fiber bundles. Also a single optical fiber bundle can be used to direct light in one side of the glass substrate or three optical fiber bundles can be used to direct light into the glass substrate. The method of illumination of the invention provides a superior signal to noise ratio as compared with conventional illumination systems.

The present invention together with the above and other objects and advantages may best be understood from the following detailed description of the preferred embodiments of the invention illustrated in the drawings, wherein:

FIG. 1A is a schematic diagram representation illustrating biochip illumination apparatus for implementing the novel biochip method of illumination in accordance with the preferred embodiment;

FIGS. 1B, 1 C, 1 D, and 1 E are detailed schematic diagram representations illustrating portions of the biochip illumination apparatus of FIG. 1A;

FIGS. 2A, 2 B, 2 C, 2 D, and 2 E are schematic diagram representations illustrating alternative biochip illumination arrangements in accordance with the preferred embodiment;

FIG. 3 is a perspective view of a glass holder for precisely positioning a glass supporting a biochip gel array used with the biochip illumination apparatus in accordance with the preferred embodiment;

FIG. 4 is a sectional view taken along line A—A of FIG. 3;

FIG. 5 is a sectional view taken along line B—B of FIG. 3; and

FIG. 6 illustrates a biochip reader including glass holder of FIG. 3 together with a positioner used with the biochip illumination apparatus in accordance with the preferred embodiment.

Having reference now to the drawings, FIG. 1 illustrates biochip illumination apparatus generally designated by the reference character 100 for implementing the novel biochip method of illumination in accordance with the preferred embodiment. Biochip illumination apparatus includes an illumination source 102 .

Illumination is provided, for example, by a low power (3-5 mW) non-collimated laser diode 102 emitting at specific wavelength such as, between 470 nm and 650 nm. Alternatively, a light emitting diode (LED) coupled with optical filter can also be used as an illumination source 102 . As an optical filter, standard filters can be used, for example: bandpass filters, longpass or shortpass barrier filters, and rejection band filters. For example, the following LED from the Newark catalog provides intensity equivalent to 5-10 mW: Super bright LED, green, 150 mcd, 50 deg, http://www.newark.com. An explanation how to translate mili-candela units to miliwatts can be found in the Basic Radiometry manual, http://www.opsci.com/technical.

As shown in FIG. 1A, a pair of fiber optic bundles 104 directs the light to opposing sides of a glass substrate 108 . The glass substrate or slide 108 supports a biochip gel array or bioarray 110 including a plurality of biochip gel pads 112 . The light is directed to the opposing sides of the glass substrate 108 by the fiber optic bundles 104 that are, for example, formed by borosilicate fiber light guides, quartz fiber light guides or plastic fiber light guides or fiber light guides formed by another suitable material.

Referring now to FIGS. 1A, 1 B and 1 C, the fiber optic bundles 104 are carried by a positioner 114 and are splayed out to make a respective fiber optic fan generally designated by the reference character 116 . The fiber optic fans 116 are one fiber thick, each defining a light line 118 or linear array of a plurality of optical fibers 120 . Each of the fiber optic bundles 104 includes a plurality of optical fibers 120 providing generally symmetrical illumination to the opposing sides of the glass substrate 108 . A single fiber 120 is illustrated in FIG. 1C that is taken along line C—C of FIG. 1 B. Each optical fiber 120 includes a polished face 122 positioned proximate to a divergent diffuser 124 . The optical fiber fans 116 define the light line 118 of the plurality of optical fiber ends or faces 122 of the respective optical fibers 120 that are received within a window 126 of the divergent diffuser 124 .

The polished optical fiber faces 122 defining the light line 118 transfer laser light to opposing sides 126 of the glass substrate 108 via the divergent diffuser 124 with only a small percentage of the laser light going back into the optical fiber 120 . This illumination process of the preferred embodiment randomizes any non-uniformity in the laser source 102 , creating a more uniform illumination source.

A thin line of light is coupled by the respective divergent diffusers 124 to illuminate the reacted bioarray 110 through a respective edge or sidewall 126 of the glass substrate 108 which diffuse light evenly and the bioarray is illuminated from the inside of the glass substrate 108 . The glass substrate 108 functions as a secondary light guide. The sidewalls 126 of the glass substrate 108 diffuse light and are not polished. Typically, commercially available glass substrate or slides 108 are not polished and do not require any additional treatment to diffuse light. The divergent diffuser 124 provides mechanical protection for the polished faces 122 of the optical fibers 120 . The divergent diffuser 124 reflects back outwardly going light to the glass 108 to increase illumination efficiency.

As shown in FIG. 1C, each optical fiber face 122 couples light through the divergent diffuser 124 at a restricted angle labeled Φ. The divergent diffuser 124 is formed of a silicon material. The divergent diffuser 124 is spaced apart from the sidewall 126 of the glass substrate 108 , for example, by 600 microns. The restricted angle Φ of light transfer is for example, 55° to 60°. Multiple arrows labeled A indicate light diffusion or mixing from each optical fiber face 122 coupled to the edge 126 of the glass substrate 108 .

Referring now to FIG. 1D, opposite fiber optic fans 116 are shown with the glass substrate 108 . As indicated by arrows labeled R, the respective centers of sequential fibers 120 of the opposite fiber optic fans 116 are offset or shifted by one radius size. This offset arrangement of the opposite fiber optic fans 116 improves light distribution within the glass substrate 108 .

Referring now to FIG. 1E, a reflector 150 can be provided to reflect outwardly going light back to the glass substrate 108 and thus avoid loss of light during illumination. One or more reflectors 150 can be provided proximate to one or more respective ends 152 or endwalls of the glass substrate 108 that are spaced apart and separate from the divergent diffusers 124 or fiber optic fans 116 . For example, the reflector 150 can be similar to the divergent diffuser 124 having generally the same restricted angle Φ of light reflection but without the open window 126 .

FIGS. 2A, 2 B, 2 C, 2 D, and 2 E illustrate alternative biochip illumination arrangements in accordance with the preferred embodiment. The same reference numbers as used with illumination apparatus 100 of FIGS. 1A and 1B are used for similar or identical components in FIGS. 2A, 2 B, 2 C, 2 D, and 2 E. As shown in FIGS. 2A, 2 B, 2 C, 2 D, and 2 E, the present invention is not limited to the use of a single pair of optical fiber bundles 116 to direct the light into opposing sides of the glass substrate 108 . A second pair of optical fiber bundle fans 116 can be provided to direct the light into opposing ends of the glass substrate 108 ; or a single optical fiber bundle fan 116 or three optical fiber bundles 116 can be provided to direct the light into the glass substrate 108 . The number of optical fiber bundle fans 116 provided depends on the size of an area of the glass substrate 108 that needs to be illuminated. In general, the entire perimeter of the glass substrate 108 including both opposing sides and both opposing ends, advantageously can be illuminated to obtain maximum uniformity.

Referring to FIG. 2A, there is shown illumination apparatus generally designated by the reference character 200 for implementing the novel biochip method of illumination in accordance with the preferred embodiment. Illumination apparatus 200 includes a pair of fiber optic fans 116 on opposing sides of glass substrate 108 with a respective light source 102 coupled to each fiber optic fan 116 .

Referring to FIG. 2B, there is shown illumination apparatus generally designated by the reference character 210 for implementing the novel biochip method of illumination in accordance with the preferred embodiment. Illumination apparatus 210 includes a first pair of fiber optic fans 116 on opposing sides of glass substrate 108 and a second pair of fiber optic fans 116 on opposing ends of glass substrate 108 with a respective light source 102 coupled to each fiber optic fan 116 .

Referring to FIG. 2C, there is shown illumination apparatus generally designated by the reference character 220 for implementing the novel biochip method of illumination in accordance with the preferred embodiment. Illumination apparatus 220 includes a pair of fiber optic fans 116 on opposing ends of glass substrate 108 and a fiber optic fan 116 on one side of glass substrate 108 with a respective light source 102 coupled to each fiber optic fan 116 .

Referring to FIG. 2D, there is shown illumination apparatus generally designated by the reference character 240 for implementing the novel biochip method of illumination in accordance with the preferred embodiment. Illumination apparatus 240 includes a first fiber optic fan 116 on one side of glass substrate 108 and a second fiber optic fan 116 on one end of glass substrate 108 with a respective light source 102 coupled to each fiber optic fan 116 .

Referring to FIG. 2E, there is shown illumination apparatus generally designated by the reference character 250 for implementing the novel biochip method of illumination in accordance with the preferred embodiment. Illumination apparatus 250 includes a single fiber optic fan 116 on one side of glass substrate 108 with a light source 102 coupled to the fiber optic fan 116 .

It should be understood that in each illumination apparatus 100 , 200 , 210 , 220 , 240 , and 250 , various low power lasers or light emitting diodes (LEDs) can be used for as the illumination source 102 . Lasers 102 having different wavelength can be used in illumination apparatus 200 , 210 , 220 , and 240 . The different wavelength lasers 102 can be used sequentially depending upon a particular target's label.

Referring now to FIGS. 3, 4 and 5 , there is shown a glass holder generally designated by the reference character 300 in accordance with the preferred embodiment for precisely positioning the glass 108 supporting multiple biochip gel arrays. Glass holder 300 includes a support housing 302 including opposing sidewalls 304 , 306 and opposing front and rear walls 308 , 310 . Glass holder includes a glass-receiving cavity 312 including a bottom surface 314 carrying a rectangular plastic springs member 316 . A Velcro material advantageously forms the plastic springs member 316 . The glass 108 sits on the plastic springs member 316 with low contact force. A pair of spring loaded rollers 320 , 322 and an axis member 324 are mounted within an upper surface 326 of the glass holder support housing 302 engaging the glass 108 with a low contact force. The glass 108 supporting the biochip gel array is easily inserted into and removed from the glass-receiving cavity 312 carried by the plastic springs member 316 . The plastic springs member 316 pushes up on the glass 108 providing an effective focal plane for reading a particular biochip gel array carried by the glass 108 . Glass holder 300 can be used with the bioarray carried by the glass substrate 108 when covered or not covered with a reaction chamber and the bioarray is provided in the focal plane of an optical system.

In accordance with features of the invention, this method of illumination provides a significantly improved illumination of the biochip gel pads 112 within the biochip array 110 . This method of illumination provides a superior signal to noise ratio as compared with conventional illumination systems.

Two variants of bioarray illumination were modeled and tested for comparison of the illumination of the invention with conventional biochip illumination. The first variant of bioarray illumination uses conventional biochip illumination where the light beams from laser hit the bioarray directly from above the biochip array. The second uses the illumination apparatus 100 of the preferred embodiment including an intermediate fiber light guide (touch-to-line) which transmits light through diffuser 122 and the side 126 of the glass substrate 108 into the glass substrate, so that the light illuminates the bioarray 110 from inside of the glass substrate. The glass substrate 108 is used as the secondary light guide in accordance with the preferred embodiment.

For both types of illumination, the uniformity was measured using fluorescent signal from empty glass. Two images was acquired: with exposure time equal to 3 seconds for the first scheme and with exposure time equal to 20 seconds for the second scheme. The exposure times were selected so that the fluorescent intensities are generally of the same order. Additional acquisition was taken for each scheme without illumination with the same exposure time to subtract the dark current of a charge coupled device (CCD) camera.

The following tables A and B contain average fluorescent signal respectively collected from small areas of image arranged into grid using conventional illumination and with illumination apparatus 100 of the preferred embodiment. The value is calculated as the sum of pixel intensities divided by the square of the area.

(For the first scheme conventional light from above the biochip array, 3 seconds, dark current subtraction):

(For the second illumination scheme in accordance with the preferred embodiment with illumination light from sides, 20 seconds, dark current subtraction):

The purpose of biochip reader is to measure fluorescent signals acquired from different pads of a microarray. Unevenness of illumination is one of the major sources of error in measurements. When the fluorescent signals are greater enough than the noise of CCD camera, the non-uniformity of illumination is the only source of error in such measurements.

Consider the relative standard deviation (RSD), defined as the standard deviation divided by the average value, is a measure of the uniformity of illumination. The RSD is the simplest criteria that reflect the quality of illumination. The uniformity of illumination is better and the error is less when the RSD is less.

On basis of data analysis the second scheme of illumination with illumination apparatus 100 of the preferred embodiment provides 3.6 times better uniformity and correspondingly 3.6 times less error than the first conventional illumination scheme.

Referring to FIG. 6, a biochip reader generally designated by the reference character 600 includes biochip illumination apparatus 100 , the glass holder 300 together with a manual positioner generally designated by the reference character 602 in accordance with the preferred embodiment. Biochip reader 600 is generally inexpensive, portable, lightweight, and simple to implement. Biochip reader 600 is illustrated in simplified form sufficient for understanding the present invention.

Biochip reader 600 includes a charge coupled device (CCD) camera 604 , a lens/lens array 606 , and a filter 608 . The CCD camera 604 and associated lens/lens array 606 and filter 608 are aligned with a window 610 in a first plate 612 . A centerline through the window 610 and the CCD camera 604 and associated lens/lens array 606 and filter 608 is labeled CL. In the biochip reader 600 , the glass holder 300 includes the plastic springs member 316 as seen in FIG. 3 that pushes up on the glass 108 with low contact forces providing an effective focal plane for reading a particular one of two biochip gel arrays 614 , 616 carried by the glass 108 . A light guide, fiber optic fan 116 is disposed proximate to the glass substrate 108 generally aligned with the window 610 . The manual positioner 602 includes a first ball bearing slide 620 coupled to a plate 618 and to the glass holder 300 and a second ball bearing slide 622 coupled to a stationary second plate 624 . The manual positioner 602 includes a base plate 626 supporting plates 612 and 624 . The manual positioner 602 includes a lever 628 extending through a slot (not shown) in the stationary second plate 624 and is mounted to the plate 618 . A plunger pin 634 also is mounted to plate 618 . A pair of stop points 630 and 632 is formed within the second plate 624 for engagement with the plunger pin 634 of the manual positioner 602 . The lever 628 is manually moved, moving the ball bearing slide 620 , plate 618 , plunger pin 634 , and the glass holder 300 and the glass substrate 108 carrying the two biochip gel arrays 614 , 616 .

As shown in FIG. 6, the plunger pin 634 is received within the stop point 630 , the biochip gel array 616 is aligned with window 610 for reading. When the lever 628 of manual positioner 602 is slidingly moved to position the plunger pin 634 with the stop point 632 , the biochip gel array 614 is aligned with window 610 for reading. Biochip reader 600 includes a case 640 and a hood 642 containing the manual positioner 602 , glass holder 300 , the CCD camera 604 and associated lens/lens array 606 and filter 608 .

In order to evaluate the use of biochip reader 600 for registration of fluorescent signals from biochips manufactured with use of different commercially available glass slides, the following experiment was carried out. Biochips containing a set of probes were produced by using slides from Motorola (3D-Link), Telechem (Superaldehyde), and Packard Bioscience (Hydrogel), as indicated in the following Tables 1 and 2. After the application of oligonucleotides bearing 5′-end amino group, the immobilization was carried out according to procedures recommended by the manufacturers.

Hybridizations with a mix of Texas Red labeled target oligonucleotides (5 fmol/μl) (Table 3) were carried for 4 h at 25° C. in 200-μl hybridization chamber (Grace Biolabs). Hybridization buffer contained 1 M guanidine isothiocyanate, 50 mM HEPES (pH 7.5), and 10 mM EDTA. After hybridization the biochips were washed for 30 sec with 6×SSPE with 0.1% Triton X-100, washed for 5 sec with MilliQ water, and dried. Hybridization signals from the biochips were recorded on the biochip reader 600 and on a commercially available scanner, Model: Bio-Chip Imager, Part No.: 902-3013001 manufactured by Packard Instrument Company, Inc., now it is Packard Bioscience.

The fluorescence intensities data was analyzed using two methods. First, the correlation function was calculated for all biochip elements of 4 chips located on slide, separately for different slide types. The correlation function is defined, for X[i] and Y[i], as M((x-Mx)(y-My))/(MxMy), where M is average of its argument array. The feature of the correlation function is that it equals 1 when and only when X and Y arrays are congruent. Since the signal is proportional to the amount of fluorescent substance, the correlation in readings between the biochip reader 600 and the scanner shows the ability of the biochip reader 600 to acquire these signals.

In the second calculation, 4 groups of biochip elements are considered: bare glass, probes that do not hybridize, probes that show hybridization with labeled target, and biochip elements with pre-immobilized labeled oligonucleotides used as markers. Correlation of average signals from all 4 types of biochip elements is also calculated for all slide types.

Table 4 summarizes data for correlation between fluorescent signals recorded by the biochip reader 600 and the scanner. For all biochips the correlation between fluorescent intensities acquired by the biochip reader 600 and the scanner is positive, and is greater than 0.9 when calculated for different types of biochip elements. This data shows the applicability of the biochip reader 600 for measurement of fluorescent signals from commercially available biochips and biochips manufactured with use of different commercially available glass slides.

While the present invention has been described with reference to the details of the embodiments of the invention shown in the drawing, these details are not intended to limit the scope of the invention as claimed in the appended claims.

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