WO2002096552A2

WO2002096552A2 - Biochip and apparatus and methods for its manufacture and use

Info

Publication number: WO2002096552A2
Application number: PCT/GB2002/002567
Authority: WO
Inventors: Aled Wynne Jones; Martin Gregory Beckett
Original assignee: Scientific Generics Limited
Priority date: 2001-06-01
Filing date: 2002-06-05
Publication date: 2002-12-05
Also published as: AU2002304518A1; WO2002096552A3; GB0113358D0

Abstract

The present invention provides a method of and apparatus for manufacturing a biochip in which different probe molecules are deposited on the substrate of the biochip with different concentrations in accordance with a predetermined spatial concentration pattern. The present invention also provides a test apparatus which uses the known concentration patterns to identify reactions of the different types of probe molecules with a test sample.

Description

BIOCHIP

This invention relates to a biochip having a number of chemical compounds attached to the surface of a substrate thereof, systems for the manufacture of such biochips and systems for their use.

Biochips are known and commonly used in the high- throughput analysis of test samples, typically new chemical or biological products, to identify, for example, potential therapeutic properties. Current biochips are manufactured using a micro-arraying device which deposits an array of different "probes" on a substrate using, for example, a multi-head positioner. The biochip is then exposed, in use, to the test sample. Each of the probes is associated with a corresponding property (such as a protein binding) and produces a detectable effect, for example an attachment to a fluorescent label which is associated with the probe, if the test sample exhibits the corresponding property. The probes may contain, for example, proteins, cells or DNA sequences . Examples of test samples that may be analysed include:

(a) proteins that give an indication of a particular disease state ("diagnostic marker");

(b) proteins or nucleic acids that give an indication of a genetic sequence variation ("genotype");

(c) small molecules that have a desired therapeutic effect ("pharmaceuticals"); or (d) a synthetic or a natural ligand that binds with high affinity ("affinity ligand" ) .

In existing biochips, as many as a thousand different probes are arrayed in respective different locations on the biochip substrate. However, prior to testing, some of the probes can become corrupted and they lose their ability to interact with the test sample, which creates uncertainty in the test results. In order to try to overcome this problem, existing techniques repeat the experiment using identical biochips to ensure that if a probe does not react with the test sample, it is not because the probe on the particular biochip being tested has become corrupt. Another solution to this problem is to provide the same probe material on two or more probe sites on the biochip. However, this reduces the number of probes that may be arrayed on the biochip and again multiple experiments have to be performed with different biochips in order to test the sample with the same number of different probes.

According to one aspect, the present invention aims to alleviate the above problems. According to one aspect, the present invention provides a method of manufacturing biochips in which the probes are laid on the surface of the biochip in a delocalised manner in accordance with a predetermined spatial concentration pattern. The concentration pattern can be any mathematically defined function, such as a sinusoidal pattern. The pattern can be continuous such as a continuous strip of probe material with a varying concentration of the probe molecules along the strip, or a discrete concentration pattern such as different concentrations of probe molecules over a number of different binding sites. Further, different probes may be located in the same location since knowledge of the different concentration patterns can be used to resolve any reaction of each of the different probes with the test sample.

Other aspects of the invention will become apparent from the following detailed description of embodiments which are described with reference to the accompanying drawings in which:

Figure 1 is a perspective view of a biochip manufacturing machine used to manufacture a biochip embodying the present invention;

Figure 2 is a block diagram illustrating a probe selection and delivery system forming part of the biochip manufacturing machine shown in Figure 1;

Figure 3 is a plan view of a biochip made by the biochip manufacturing machine shown in Figure 1;

Figure 4 is a block diagram illustrating the main components of a biochip testing machine;

Figure 5 is a block diagram illustrating the main components of an image processing unit forming part of the testing machine shown in Figure 4; Figure 6a is a plot illustrating a first intensity profile for a first probe determined by an intensity profile determining unit forming part of the image processing unit shown in Figure 5;

Figure 6b is a plot illustrating a second intensity profile for a second probe determined by an intensity profile determining unit forming part of the image processing unit shown in Figure 5;

Figure 7 is a block diagram illustrating the main components of a probe selection and delivery system forming part of a biochip manufacturing machine according to a second embodiment;

Figure 8 is a plan view of a biochip made by the biochip manufacturing machine of the second embodiment;

Figure 9 schematically illustrates the way in which a biochip having a number of different probe concentration patterns may be formed on the surface of a biochip; and

Figure 10 is a flowchart showing a typical drug development process .

FIRST EMBODIMENT

Micro-Arraying Apparatus

Figure 1 is a perspective view of a micro-arraying apparatus 1 having a base 3 which is positioned on a table 5 via four anti-vibration feet, three of which are visible in Figure 1 and referenced 5a to 5c. In use, a shield (not shown) also surrounds the apparatus 1, to prevent vibrations caused by air currents .

A worktop 7 is mounted on an x-direction translation stage 9, which is in turn mounted on a y-direction translation stage 11 that is mounted on the base 3. A head device having a housing 13 is mounted on a z- direction translation stage 15, which in turn is mounted on the base 3. As shown, each of the translation stages 9, 11 and 15 moves along a respective bearing slide 17a, 17b, 17c having a dove-tail profile. The translation stages are driven along the bearing slide 17 by respective motorised lead screws (not shown) . As shown in Figure 1, a dispensing nozzle 21 is provided on the underside of the housing 13, and is used to dispense probe material onto the substrate of a biochip 19.

In this embodiment, a control circuit (shown in Figure 2) within the housing 13 controls the dispensing of probes from the dispensing nozzle 21 onto the biochip 19 so that the probes are laid onto the substrate of the biochip 19 in accordance with a predetermined concentration pattern. Figure 2 shows in more detail the components of this control circuit and the contents of the housing 13. As shown, in this embodiment, the housing 13 includes a number (N) of probe reservoirs labelled 31-1 to 31-N, each containing a different probe in a solvent at a maximum required concentration. As shown, these reservoirs 31 feed into a selector 3.3 which operates to select a probe from one or more of the reservoirs 31. In this embodiment, the particular probes that are selected at a given time are controlled by a control unit 35 which also controls the position of the dispensing nozzle 21 relative to the biochip 19 by outputting appropriate control signals to an x, y, z position control circuit 37 which in turn controls the x, y, z actuators 38 used to move the worktop 7 relative to the dispensing nozzle 21. The control unit 35 can also control the concentration of each of the probes applied to the biochip 19 by controlling the addition of extra solvent to the selected probe from a solvent reservoir 41. The original probe and solvent is then mixed with the additional solvent in a mixer 43 before being output onto the biochip 19 via the dispensing nozzle 21.

In this embodiment, the control unit 35 controls the selection of the probes, their concentration and the position of the dispensing nozzle 21 relative to the biochip 19 in accordance with predetermined concentration pattern data 39, which defines the required concentration pattern for each probe to be placed on the microchip 19. In this embodiment, the control unit 35 controls the dispensing of the probes onto the biochip 19 so that the biochip 19 has an array of discrete binding sites, with each binding site having three different probes, and with each probe being provided in three different binding sites at a respective different concentration level. Figure 3 is a plan view of a biochip 19 showing in more detail the different binding sites 108 formed on the surface 302 of the biochip 19. As shown, in this embodiment, the binding sites 108 are arrayed in a regular two-dimensional array. Further, in this embodiment, the concentration pattern data 39 ensures that the same three probes are provided at different levels of concentration within three adjacent probe sites 108. For example, for the three probe sites 108-1, 108-2 and 108-3, three different probes (probes A, B and C) at varying concentrations are provided on these sites. In this embodiment, probe site 108-1 contains 25% concentration of probe A, 75% concentration of probe B and 100% concentration of probe C; probe site 108-2 contains 50% concentration of probe A, 50% concentration of probe B and 50% concentration of probe C; and probe site 108-3 contains 75% concentration of probe A, 25% concentration of probe B and 50% concentration of probe C. A similar variation of the probe concentrations is applied to the other probes in the other probe sites 108 shown in Figure 3.

As shown in Figure 3, the biochip 19 also includes two fiducial marks 305a and 305b which can be used as position references for locating the different probe sites 108 on the biochip 19. Additionally, in order that the subsequent testing apparatus (not shown) can identify the different probes on the biochip 19, each biochip 19 is also provided with a unique serial number 306 that is marked on the substrate 302. In this embodiment, the serial number is provided both in human readable form 306a and in machine-readable form 306b. In this embodiment, the format of the data 306b is such that the testing apparatus can readily determine the serial number of the biochip 19 through suitable image processing. The concentration pattern data 39 used to control the depositing of the different probes onto the biochip 19 is also associated with the serial number and sold with the biochip 19. As will be described in more detail below, this concentration pattern data is required by the testing apparatus to be able to resolve the different reactions by the different probes.

Test Apparatus The test apparatus used in this embodiment is similar to the arraying apparatus shown in Figure 1 except the sample to be tested is dispensed from the dispensing nozzle 21 onto the probe sites 108 of the biochip 19. In addition, an illumination source and a CCD camera are provided to illuminate the biochip 19 after the test sample has been provided on the probe sites 108 and to subsequently record the fluorescence of the different probe sites as they react with the test sample under the excitation caused by the illumination source.

Figure 4 is a block diagram illustrating the main components of the test apparatus 400 used in this embodiment. As shown, the test apparatus 400 includes a controller 401 which controls the dispensing of the test sample from a test sample reservoir 403 via the dispensing nozzle 21, under control of software instructions (not shown) stored within the controller 401. The controller 401 is also arranged to output appropriate control signals to a lamp driver 402 for turning the lamp 300 on and off. After the test sample has been applied to the biochip 19 and the lamp 300 has been activated, image data from the CCD camera 304 is processed by an image processing unit 404 to determine how each of the different probes in each of the probe sites 108 fluoresce in response to the light from the lamp 300. The image processing unit 404 does this using the information about what probes are located in which probe sites and at what concentrations (i.e. from the concentration pattern data 39). This pattern data 39 is input to the controller 401 via an appropriate data interface 405 such as a floppy disc reader. The controller 401 then passes this concentration pattern data to the image processing unit 404 for use in analysing the image data received from the camera 304. The intensity profiles determined by the image processing unit 404 are then passed to the controller 401 which processes them to make quantitative measures of the reaction of the test sample with the probes. Various known ways are used for making these quantitative measures. The measures most commonly used include the steepest gradient of the profile(s) and the peak intensity of the profile(s) relative to a noise floor. These quantitative measures are then output to a user via a user interface 406 such as a CRT display. Jmage Processing Unit

Figure 5 is a block diagram illustrating the main components of the image processing unit 404 used in this embodiment. As shown, the CCD pixel image data generated by the CCD camera 304 for a current time frame is input to a probe area locator 421 which processes the image data to identify the location of the different probe sites 108 on the biochip 19. In this embodiment, the probe area locator 41 initially identifies which pixel values of the CCD pixel data correspond to the different probe sites. It does this by processing the initial CCD image to identify the pixels corresponding to the fiducial marks 305. The probe locator 41 then uses x-y position data which identifies the position of each probe site 108 relative to the fiducial marks 305 to determine the pixels which correspond to the probe sites. In this embodiment, this x-y position data for the different probe sites 108 forms part of the concentration pattern data 39 provided with the biochip 19. As those skilled in the art will appreciate, once the probe area locator 41 has determined the pixels corresponding to the probe sites 108, it does not need to redetermine these pixels for subsequent image frames, provided the biochip 19 remains fixed relative to the CCD camera 304.

The pixel values associated with the probe sites are then passed to a relative intensity determining unit 423 which determines the relative intensity between these pixel values and pixel values that are not associated with the probe sites. The relative intensity determining unit 423 therefore effectively normalises the pixel intensity values generated from the probe sites 108 to take into account the background luminescence or noise caused by random attachment of the test sample to the surface of the biochip 19. The normalised pixel values for each probe site are then passed to a pattern matching unit 425 which uses the concentration pattern data 39 received from the controller 401 to determine the intensity value for the different probes in the current CCD image. The intensity values determined by the pattern matching unit 425 are then passed to the intensity profile determining unit 427 which monitors the intensity values generated for each of the different probes in successive CCD images, to determine an intensity profile for each of the probes showing how the fluorescence of the probe changes with time.

Figures 6a and 6b schematically illustrate two different intensity profiles that may be generated by the intensity profile determining unit 427. In particular, the intensity profile 431 shown in Figure 6a represents the intensity profile determined for probe A. As shown, the intensity profile 431 is determined from the intensity values obtained from successive CCD images, which are represented by the height of the different arrows (e.g. A₀, A₅, A₉) underneath the intensity profile 431. Similarly, Figure 6b shows the intensity profile 433 determined for probe B. As can be seen from comparing the intensity profiles shown in Figure 6, probe A is slower to react with the test sample but fluoresces more strongly than probe B. These intensity profiles are then passed to the controller 401 which performs the analysis described above.

Pattern Matching Unit

As described above, in this embodiment, each probe is disposed on three consecutive probe sites 108 with different concentrations of the probe in each of those three sites. The pattern matching unit 425 then uses the known relative concentrations of the three different probes in the three different probe sites 108 to determine the intensity associated with each probe. In this embodiment, with the above concentrations, this involves solving the following equations:

0.25Ai + 0.75Bi + Cj_. = I,.¹ 0.5Ai + 0.5Bi + 0.5Ci = l ² 0.75Ai + 0.25Bi + 0.5Cj_. = Ij_. ³

where A_± is the intensity associated with probe A in CCD frame i; B_A is the intensity associated with probe B in CCD frame i; Ci is the intensity associated with probe C in CCD frame i; Ij_. ¹ is the value of the pixel corresponding to the first of the three probe sites 108-1 with the three probes A, B, C in CCD frame i; I_± ² is the value of the pixel corresponding to the second of the three probe sites 108-2 with the three probes A, B, C in CCD frame i; and Ij³ is the value of the pixel corresponding to the second of the three probe sites 108- 3 with the three probes A, B, C in CCD frame i. In this embodiment, the resolution of the CCD camera 304 is such that each of the probe sites corresponds to a number of pixels and the CCD values of the pixels associated with a probe site are averaged to generate the l intensity values in the above equation.

As those skilled in the art will appreciate, the above equation can be solved for each of the three unknown intensity values (Ai, B_if C_A) for the three different probes. However, as those skilled in the art will appreciate, the relative concentrations of each of the probes in the three probe sites 108 must be carefully chosen so that none of the three equations is a multiple of the other two equations. The pattern matching unit 425 solves these equations for all of the different groups of probes on the biochip 19.

SECOND EMBODIMENT

In the first embodiment described above, the arraying machine deposited the probes in discrete probe sites 108 on the biochip 19. Alternatively, the probes may be deposited on the surface of the biochip 19 as continuous strips of probe with different concentrations. Figure

7 is a block diagram illustrating the main components of a dispensing unit forming part of a biochip manufacturing machine used in a second embodiment. In this embodiment, the biochip manufacturing machine is arranged to deposit a continuous strip of each probe material on the surface of the biochip 19, with the concentration of probe material in each strip varying along the length of the strip. The components which are the same as those used in the first embodiment have been labelled with the same reference numeral and will not be described again.

As shown in Figure 7, N probe reservoirs 31-1 to 31-N are provided, each having a solution of a respective different probe and solvent at the highest . level of concentration required. As shown, these reservoirs are connected through a switch 501 to a valve 503. The particular probe reservoir connected to the valve 503 through the switch 501 is controlled by the control unit 35 in accordance with the concentration pattern data 39 ' . The control unit 35 also controls the valve 53 to add a required amount of additional solvent from the solvent reservoir 41 to the probe being dispensed. Again, the amount of the additional solvent is controlled by the control unit 35 in dependence upon the concentration pattern data 39'. The probe and additional solvent are then output onto the biochip 19 via the dispensing head 21. By varying the amount of additional solvent added to the probe material in accordance with . the concentration pattern data 39', the dispensing unit can generate strips of probe material having a concentration pattern along the length, of the strip.

Figure 8 is a plan view schematically illustrating the different strips 551-1 to 551-N of probe material deposited on the biochip 19. As represented by the varying density of dots along the length of the strips 551, the concentration of the probe material is made to vary in accordance with a predetermined pattern along the length of the strip 551. In this embodiment, the pattern of variation of the probes in each of the different strips 551 are different (either in spatial frequency or phase). Although this is not essential, it allows the identification of the different probes from the different patterns of probe material on the biochip 19. As shown in Figure 8, in this embodiment, the concentration pattern of each probe in a strip 551 is arranged to increase and decrease at a predetermined frequency. As discussed above, this continuous variation of the probe concentration along the strip 551 is controlled by the addition of varying amounts of additional solvent from the solvent reservoir 41 in accordance with the concentration pattern data 39'. As those skilled in the art will appreciate, it is not essential to continuously vary the probe concentration along the length of the strip 551. A more digital variation of the concentration pattern may be performed. This is illustrated in strip 551-N, where the concentration of the probe varies between two different concentration levels along the length of the strip 551-N.

In this embodiment, the testing apparatus used to test the biochip 19 is structurally the same as the testing apparatus used in the first embodiment and described above with reference to Figures 4 and 5. The only difference is in the operation of the pattern matching unit 425 which, instead of solving discrete simultaneous equations, correlates the CCD values with predetermined mathematical functions defining the way in which the concentration pattern varies along the length of the strips 551. In this embodiment, the mathematical equations are simple sinusoids, whose frequency matches the spatial frequency of variation of the probe concentration along the strip 551. Further, as mentioned above, in this embodiment it is not essential for the testing apparatus to know which probe has been laid in each of the strips 551 of probe material, since the concentration pattern data 39 provided with the biochip 19 identifies, for each of the different probe materials, the spatial pattern of that probe within the corresponding strip 551 of probe material.

THIRD EMBODIMENT

In the second embodiment described above, the biochip manufacturing machine was arranged to output varying concentration of each probe material onto the surface of the biochip 19. This allowed the deposition of the different probe materials in accordance with different concentration patterns on the biochip 19. In this third embodiment, the probe dispensing unit is arranged to apply an electric charge to the different probes and then to deposit the probes in strips 551 across the biochip 19 with a constant concentration. The concentration pattern of each probe along the strip 551 is then varied by applying an electric field at an appropriate wavelength along the length of the strip 551 of probe material. As a result, the charged probe molecules migrate in accordance with the spatial frequency, of the electric field.

Figure 9 schematically illustrates the way in which the biochip 19 shown in Figure 8 can be made using charged probe materials and electric fields. As shown, two electrodes are provided at each end of each strip 511 of probe material. These electrodes are labelled 601-a, 601-b to 609-a, 609-b for the strips 511 of probe material shown. The different electric fields generated between these electrodes are represented at 602, 604, 606, 608 (which are single tone fields) and 610 (which is a square wave field) . As shown, at the peak of the electric fields, the concentration of the probe material is at its greatest whereas at the lowest point of the electric fields, the concentration of the probe material is at its lowest. This is because, the electric charge applied to the probe materials is such as to attract it to the electric field generated by the electrodes. As can be seen from Figure 9, the electric field applied to each strip 511 of probe material is different either in its frequency or its phase. Again, this is used in this embodiment in order to uniquely label each of the different probes, so that the testing apparatus can identify which probe or probes react with the test sample.

In this embodiment, in order to ensure that the probe material binds to the surface of the substrate with the required concentration pattern, after the strip of probe material 511 has been deposited on the biochip 19_.and the appropriate electric field has been applied along the length of the strip 511, the solvent is dried leaving just the probe material bound to the surface of the biochip 19. At this point, the electric field can be removed and the next strip 511 of probe material can be deposited on the substrate. In this embodiment, it is preferable to deposit each probe onto the biochip 19 one at a time, so that the electric fields used to create the concentration patterns along the strips 511 of probe material, do not interfere with each other. However, this is not essential.

DRUG DEVELOPMENT PROCESS

Figure 10 is a flowchart illustrating a typical drug development process that uses the biochips manufactured in the above embodiments. Firstly, in step si, a new sample is prepared and then the new sample is tested, in step s3, for potential therapeutic properties. In this embodiment, the testing for potential therapeutic properties includes the step of using a biochip made in accordance with any of the above embodiments and using the corresponding test machine described in those embodiments .

If no potential therapeutic property is discovered, the process ends, whereas if a potentially therapeutic property is discovered, clinical drug development is performed in step s5. There are many reasons why a drug entering clinical development does not result in a commercial product. For example, the drug could have an adverse side effect or could be prohibitively expensive to manufacture commercially. If the new samples do not satisfy, in step s7, the requirements for commercial manufacture, then the drug development process ends. If, however, the clinical drug development is successful, then a drug is commercially manufactured in step s9.

As those skilled in the art will appreciate, analogous development process will be performed for biological products.

MODIFICATIONS AND ALTERNATIVE EMBODIMENTS

A number of modifications and alternatives to the above embodiments will now be described. In the above embodiments, a test sample was deposited on the biochip under test from a dispensing nozzle. The test sample may be deposited on each probe site individually or the test sample may be applied more liberally across the entire surface of the biochip.

In the above embodiments, a light source was used to excite the probe array. Various different sources of radiation may be provided, such as: incandescent light sources, LEDs, lasers, chemical light sources, radioactive radiation sources, x-ray sources; luminescent sources etc. The radiation generated by these sources may be directly directed towards the probe sites or indirectly via a beam splitter, a scanning optic, spatial light modulators, a lens array, optical fibres, an optical waveguide (solid or liquid) etc. In the above embodiment, a CCD camera was used to detect the fluorescence of the probes interacting with the test sample. As those skilled in the art will appreciate, other light detectors may be used, such as a photodiode, a phototransistor, a photovoltaic detector etc.

In the second and third embodiments described above, separate strips of probe material were deposited on the substrate of the biochip. In an alternative embodiment, the strips of biochip material may be deposited in an overlapping manner on the biochip, provided the different concentrations of probes that are to be overlaid do not interact with each other in a way that would disturb the binding with the target. This is similarly the case for the first embodiment, where multiple probes were applied to discrete binding sites. If the probes would interact with each other, then it is possible to create sub-sites within a binding site, where the different probes can be located. Provided the imaging equipment can then integrate the sub-sites so that they appear to be one binding site, the same processing can be performed. This can be achieved either by ensuring that one CCD pixel corresponds to one binding site, or where multiple pixels are provided for each binding site, by integrating the pixel values for each binding site.

In the first embodiment described above, the probes were deposited onto the surface of the biochip in groups of three onto three different binding sites . This allowed the testing system to form three simultaneous equations from which it could determine the intensity of fluorescence of each of the individual probes . As those skilled in the art will appreciate, it is not necessary to have three probes in each group. Groups of n probes may be processed together, provided they are present in varying concentrations in m probe sites, where m is greater than or equal to n. However, as those skilled in the art will appreciate, there are advantages in ensuring that m is greater than n, since this allows m-n of the probe sites to become corrupt either as a result of noise or as a result of the probe molecules within these probe sites becoming inactive, whilst still allowing for the determination of the reaction of each probe to the test sample. This is because, with greater than n, more simultaneous equations can be obtained than there are unknowns. This also allows consistency checking and statistical analysis to be carried out. As those skilled in the art will appreciate, the concentrations of the different probes in each probe site should be chosen to obtain n independent simultaneous equations with the remaining,m-n simultaneous equations being arranged so that they are totally dependent on the n independent simultaneous equations. In this way, if one or more of the probe sites becomes corrupt, the information from the other sites can be used to resolve this ambiguity. This may be achieved by performing different summations or subtractions of the n independent simultaneous equations . For example, in the first embodiment described above, another probe site may be provided with 75% concentration of probe A, 75% concentration of probe B and 100% concentration of probe C. This would yield the following simultaneous equation for that probe site:

As those skilled in the art will appreciate, this equation is derived by summing the three equations given in the first embodiment and then dividing the concentrations by two.

In the first embodiment described above, the probes were deposited onto the surface of the biochip in groups of three, with three different binding sites in each group. As those skilled in the art will appreciate, the biochip may be arranged with different numbers of probes and binding sites in each group. However, such an embodiment is not preferred since it over complicates the amount of concentration pattern data required and the processing required in the subsequent test appratus.

In the second and third embodiments described above, the different probes on the biochip had different concentration patterns. As those skilled in the art will appreciate, this is not essential. The concentration pattern for two or more of the probes may be the same. In this case, positional information about which probes are located in which locations on the biochip will have to be provided with the biochip, so that the testing apparatus can distinguish between the probes having the same concentration pattern.

In the second and third embodiments described above, "one dimensional" strips of probe material were deposited on the surface of the biochip. As those skilled in the art will appreciate, two dimensional strips of the different probes may be deposited on the biochip surface, with the different strips of probe material overlapping and with the probe molecules within each two dimensional strip having a respective two dimensional concentration pattern.

In the third embodiment described above, the probes were charged with an electric charge and then an electric field was used to vary the probe concentration pattern on the biochip. As those skilled in the art will appreciate, a similar result can be achieved using acoustic waves to vary the concentration pattern of the probes on the biochip.

In the above embodiments, the probes were deposited onto the biochip from a single dispensing nozzle. As those skilled in the art will appreciate, arrays of dispensing nozzles may be used, with each dispensing nozzle being arranged to dispense either a different probe or the same probe but with a different concentration.

In the above embodiments, the test apparatus determined intensity profiles for each of the different probes on the biochip. As those skilled in the art will appreciate, instead of determining the intensity profile, the testing apparatus may integrate the intensity over time to determine solely if there has been any reaction with the test sample.

In the above embodiments, the biochip was provided with a serial number in both human readable form and in machine readable form. The machine readable form was implemented as a two-dimensional barcode on the surface of the substrate. In alternative embodiments the biochip substrate may be provided with a magnetic strip on which the serial number of the biochip may be recorded. The serial number may be either pre-recorded onto a blank substrate and read by the biochip manufacturing machine or the biochip manufacturing machine may record the serial number onto the magnetic strip during the manufacture of the biochip. By providing the magnetic strip with a sufficient data capacity, the identities, and locations on the biochip, of the deposited probes may be recorded onto the biochip itself. As an alternative to a magnetic strip, a radiofrequency (RF) tag may be used to receive and store data on the biochip. An RF tag uses an integrated circuit and a non-contact mechanism to read and write data to and from the RF tag. Typically, an alternating magnetic field is used to both transmit data to the RF tag and to provide a power source for the RF tag and the RF tag transmits data by modulating the alternating magnetic field.

The substrate from which a biochip is manufactured may be glass, ceramic, plastic, metal or a living tissue.

Various other alternatives will be apparent to those skilled in the art and will not be described here.

Claims

CLAIMS :

1. A method of manufacturing a biochip comprising the steps of: providing a substrate onto which different types of probe molecules can be deposited; and attaching probe molecules of the same type in different concentrations on the substrate in a delocalised manner in accordance with a predetermined spatial concentration pattern.

2. A method according to claim 1, wherein said attaching step attaches the same type of probe molecules onto a plurality of discrete probe sites on the surface of said substrate.

3. A method according to claim 2, wherein said attaching step attaches different types of probe molecules on the substrate in a respective predetermined spatial concentration pattern.

4. A method according to claim 3, wherein said attaching step attaches groups of n different types of probe molecules onto m discrete probe sites on the surface of said substrate, where n is less than or equal to m.

5. A method according to claim 4 , wherein said attaching step attaches different types of probe molecules in a plurality of said groups on said substrate, and wherein n and m are the same in each group.

6. A method according to claim 4 or 5, wherein said attaching step attaches different concentrations of probe molecules in each probe site.

7. A method according to claim 1, wherein said attaching step attaches the same probe molecules in a substantially continuous pattern on said substrate.

8. A method according to claim 7, wherein said attaching step attaches the same probe molecules in a substantially continuous one dimensional pattern on said substrate.

9. A method according to claim 7, wherein said attaching step attaches the same probe molecules in a substantially continuous two dimensional pattern on said substrate.

10. A method according to any of claims 7 to 9, wherein said attaching step attaches different probe molecules in diff rent concentrations in a respective substantially continuous pattern on said substrate in accordance with a respective predetermined spatial concentration pattern.

11. A method according to any of claims 7 to 10, further comprising the step of depositing said probe molecules on said substrate in said substantially continuous pattern .

12. A method according to claim 8, further comprising the step of dynamically varying the concentration of the probe material in accordance with said predetermined spatial concentration pattern during the depositing step.

13. A method according to claim 11, wherein said depositing step deposits said probe molecules on said substrate with a uniform concentration and wherein the method further comprises the step of causing the migration of probe molecules within said pattern in accordance with said predetermined spatial concentration pattern.

14. A method according to claim 13, comprising the step of electrically charging the probe molecules prior to said depositing step and wherein said causing step uses an electric field having a predetermined spatial pattern to cause said migration of said probe molecules.

15. A method according to claim 13, wherein said causing step uses pressure waves having a predetermined spatial pattern to cause said migration of said probe molecules.

16. A method according to any of claims 11 to 15, wherein said probe molecules are carried in a liquid, a gas or a gel and wherein said depositing step deposits the liquid, gas or gel carrying the probe onto the substrate.

17. A method according to any preceding claim, wherein the probe molecules comprise at least one of: a chemical, a protein, a sequence of DNA, a bacterium, a virus and a cell.

18. A method according to any preceding claim, wherein said attaching step attaches a plurality of different probe molecules onto said substrate at different times.

19. A method according to any preceding claim, further comprising the step of storing data defining the predetermined spatial concentration pattern with which probe molecules are attached to said substrate.

20. A method according to claim 19, wherein said substrate carries a fiducial mark and further comprising the step of storing data identifying the position of said probes on said substrate relative to said fiducial mark.

21. A method according to claim 19 or 20, further comprising the step of outputting said stored data on a carrier medium.

22. A method according to any preceding claim, wherein said substrate carries a serial number and further comprising the step of reading the serial number of the substrate.

23. A method according to any of claims 1 to 21, further comprising the step of generating a serial number and recording the serial number on the substrate.

24. A method according to claim 22 or 23, further comprising the step of associating the identity of the different probe molecules attached to the substrate with the serial number of the substrate.

25. A method according to any of claims 22 to 24, when dependent on any of claims 19 to 21 further comprising the step of associating said stored data with the serial number of the substrate.

26. A biochip manufacturing apparatus comprising: means for receiving a substrate onto which different types of probe molecules can be deposited; and means for attaching probe molecules of the same type in different concentrations on the substrate in a delocalised manner in accordance with a predetermined spatial concentration pattern.

27. An apparatus according to claim 26, wherein said attaching means is operable to attach the same type of probe molecules onto a plurality of discrete probe sites on the surface of said substrate.

28. An apparatus according to claim 27, wherein said attaching means is operable to attach different types of probe molecules on the substrate in a respective predetermined spatial concentration pattern.

29. An apparatus according to claim 28, wherein said attaching means is operable to attach groups of n different types of probe molecules onto m discrete probe sites on the surface of said substrate, where n is less than or equal to m.

30. An apparatus according to claim 29, wherein said attaching means is operable to attach different types of probe molecules in a plurality of said groups on said substrate, and wherein n and m are the same in each group.

31. An apparatus according to claim 29 or 30, wherein said attaching means is operable to attach different concentrations of probe molecules in each probe site.

32. An apparatus according to claim 26, wherein said attaching means is operable to attach the same probe molecules in a substantially continuous pattern on said substrate.

33. An apparatus according to claim 32, wherein said attaching means is operable to attach the same probe molecules in a substantially continuous one dimensional pattern on said substrate.

34. An apparatus according to claim 32, wherein said attaching means is operable to attach the same probe molecules in a substantially continuous two dimensional pattern on said substrate.

35. An apparatus according to any of claims 32 to 34, wherein said attaching means is operable to attach different probe molecules in different concentrations in a respective substantially continuous pattern on said substrate in accordance with a respective predetermined spatial concentration pattern.

36. An apparatus according to any of claims 32 to 35, further comprising means for depositing said probe molecules on said substrate in said substantially continuous pattern.

37. An apparatus according to claim 33, further comprising means for dynamically varying the concentration of the probe material to be deposited by said depositing means in accordance with said predetermined spatial concentration pattern.

38. An apparatus according to claim 36, wherein said depositing means is operable to deposit said probe molecules on said substrate with a uniform concentration and further comprising means for causing the migration of probe molecules within said pattern in accordance with said predetermined spatial concentration pattern.

39. An apparatus according to claim 38, comprising means for electrically charging the probe molecules prior to or during deposition by said depositing means and wherein said means for causing comprises means for generating an electric field having a predetermined spatial pattern for causing said migration of said probe molecules.

40. An apparatus according to claim 38, wherein said causing means comprises means for generating pressure waves having a predetermined spatial pattern for causing said migration of said probe molecules.

41. An apparatus according to any of claims 36 to 40, wherein said probe molecules are carried in a liquid, gas or gel and wherein said depositing means is operable to deposit the liquid, gas or gel carrying the probe onto the substrate.

42. An apparatus according to any of claims 26 to 41, wherein the probe molecules comprise at least one of: a chemical, a protein, a sequence of DNA, a bacterium, a virus and a cell.

43. An apparatus according to any of claims 26 to 42, wherein said attaching means is operable to attach a plurality of different probe molecules onto said substrate at different times.

44. An apparatus according to any of claims 26 to 43, further comprising means for storing data defining the predetermined spatial concentration pattern with which probe molecules are attached to said substrate.

45. An apparatus according to claim 44, wherein said substrate carries a fiducial mark and further comprising means for storing data identifying the position of said probes on said substrate relative to said fiducial mark.

46. An apparatus according to claim 44 or 45, further comprising means for outputting said stored data on a carrier medium.

47. An apparatus according to any of claims 26 to 46, wherein said substrate carries a serial number and further comprising means for reading the serial number of the substrate.

48. An apparatus according to any of claims 26 to 46, further comprising means for generating a serial number and means for recording the serial number on the substrate.

49. An apparatus according to claim 47 or 48, further comprising means for associating the identity of the different probe molecules attached to the substrate with the serial number of the substrate.

50. An apparatus according to any of claims 47 to 49, when dependent on any of claims 44 to 46 further comprising the step of associating said stored data with the serial number of the substrate.

51. A biochip comprising: a substrate; and a plurality of probe molecules of the same type arranged in different concentrations on the substrate in accordance with a predetermined spatial concentration pattern.

52. A biochip according to claim 51, wherein said probe molecules are distributed over a plurality of discrete probe sites on the surface of said substrate.

53. A biochip according to claim 52, comprising plural types of probe molecules on said substrate on a respective predetermined spatial concentration pattern.

54. A biochip according to claim 53, wherein said plurality of different probe molecules are arranged in groups of n different types of probe molecules in m discrete probe sites on the surface of said substrate, where n is less than or equal to m.

55. A biochip according to claim 54, wherein said different types of probe molecules are attached in a plurality of said groups on said substrate, and wherein n and m are the same in each group.

56. A biochip according to claim 54 or 55, wherein different concentrations of probe molecules are provided in each probe site.

57. A biochip according to claim 51, wherein said probe molecules are attached to said biochip in a substantially continuous pattern on said substrate.

58. A biochip according to claim 57, wherein the same probe molecules are arranged in a substantially continuous one dimensional pattern on said substrate.

59. A biochip according to claim 57, wherein the same probe molecules are arranged in a substantially continuous two dimensional pattern on said substrate.

60. A biochip according to any of claims 57 to 59, wherein different probe molecules are attached to said substrate in different concentrations in a respective substantially continuous pattern on said substrate in accordance with a respective predetermined spatial concentration pattern.

61. A biochip according to any of claims 51 to 60, wherein the probe molecules comprise at least one of: a chemical, a protein, a sequence of DNA, a bacterium, a virus and a cell.

62. A biochip according to any of claims 51 to 61, comprising a fiducial mark on said substrate.

63. A biochip according to any of claims 51 to 62, further comprising a serial number on said substrate.

64. In combination a biochip according to any of claims 51 to 63, and position data identifying the position of said probe molecules on said substrate.

65. An apparatus for testing a biochip having a plurality of probe molecules of the same type arranged in different concentrations on the substrate in accordance with a predetermined spatial concentration pattern, the apparatus comprising: means for receiving data defining the predetermined spatial concentration pattern of the probe molecules on the biochip; means for applying a test sample to said biochip; means for sensing an interaction of the test sample with the probes and for outputting a signal that varies with the interaction; and means for processing the output signal and the received data to determine one or more parameters indicative of the interaction of the test sample with the probes .

66. An apparatus according to claim 65, wherein said sensor is operable for sensing light emitted as a result of the interaction of the test sample with the probe molecules .

67. An apparatus according to claim 65 or 66, wherein a plurality of different types of probe molecules are provided on said substrate in a respective spatial concentration pattern, and wherein said processing means is operable to deconvolve reactions of the test sample with one or more of the different probe molecules using the received data defining the predetermined spatial concentration patterns of the respective probe molecules.

68. An apparatus according to claim 67, wherein said different types of probe material are provided in discrete probe sites on the surface of said biochip, with each probe site including different types of probe molecules at different concentration levels and wherein said processing means is operable to formulate and solve simultaneous equations defined by the probe molecules which are present in each probe site and the levels of concentration of each probe molecule in each probe site.

69. An apparatus according to any of claims 65 to 68, wherein said processing means is operable to derive a temporal interaction profile identifying how the interaction between the test sample and the probes varies with time.

70. An apparatus according to any of claims 65 to 69, wherein said biochip comprises a biochip according to any of claims 51 to 63.

71. A method of testing a biochip having a plurality of probe molecules of the same type arranged in different concentrations on the substrate in accordance with a predetermined spatial concentration pattern, the method comprising the steps of: receiving data defining the predetermined spatial concentration pattern of the probe molecules on the biochip; applying a test sample to said biochip; sensing an interaction of the test sample with the probes and outputting a signal that varies with the interaction; and processing the output signal and the received data to determine one or more parameters indicative of the interaction of the test sample with the probes.

72. A method of manufacturing a biological or chemical product comprising the steps of: testing a plurality of biological or chemical samples to identify a test sample having a therapeutic property; developing a biological or chemical product from the identified test sample; and commercially manufacturing the biological or chemical product; wherein the testing of the test sample comprises the steps of: manufacturing a biochip using the method of any of claims 1 to 25, the probe being associated with a corresponding property and producing a detectable effect when exposed to a chemical or biological sample exhibiting the associated property; exposing the biochip to each of said test samples; and for each of the plurality of test samples, monitoring for the presence of the respective properties associated with the probe.

73. A process of producing a biological or chemical product comprising the steps of: testing a plurality of biological or chemical samples to identify a test sample having a therapeutic property; developing a biological or chemical product from the identified test sample; and commercially manufacturing the biological or chemical product; wherein the testing of the test samples comprises the steps of: testing a biochip using a testing apparatus according to any of claims 65 to 70.

74. A computer readable medium storing computer executable instructions for controlling a programmable computer device to carry out the method of any of claims 1 to 25 or the method of claim 71.