U.S. patent application number 10/727566 was filed with the patent office on 2004-09-09 for support for analyte determination methods and method for producing the support.
This patent application is currently assigned to FeBiT Ferrarius Biotechnology GmbH. Invention is credited to Lindner, Hans, Miller, Manfred, Stahler, Cord F., Stahler, Fritz, Stahler, Peer F..
Application Number | 20040175734 10/727566 |
Document ID | / |
Family ID | 27512654 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040175734 |
Kind Code |
A1 |
Stahler, Cord F. ; et
al. |
September 9, 2004 |
Support for analyte determination methods and method for producing
the support
Abstract
A method for producing a support for determining analytes. The
method comprises the steps of (a) providing a support comprising at
least one channel, comprising a conduit having an intake and an
outlet for passing fluid from the intake to the outlet, in the
support body, (b) passing liquid with building blocks for
synthesizing polymeric receptors through the channel or channels of
the support body, (c) site- and/or time-specifically immobilizing
the receptor building blocks in each case on predetermined
positions in the channel or channels by illumination and (d)
repeating steps (b) and (c) until the required receptors have been
synthesized in each case on the predetermined positions.
Inventors: |
Stahler, Cord F.; (Weinheim,
DE) ; Stahler, Peer F.; (Mannheim, DE) ;
Miller, Manfred; (Munchen, DE) ; Stahler, Fritz;
(Weinheim, DE) ; Lindner, Hans; (Stuttgart,
DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
FeBiT Ferrarius Biotechnology
GmbH
Mannheim
DE
|
Family ID: |
27512654 |
Appl. No.: |
10/727566 |
Filed: |
December 5, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10727566 |
Dec 5, 2003 |
|
|
|
09763914 |
May 11, 2001 |
|
|
|
09763914 |
May 11, 2001 |
|
|
|
PCT/EP99/06317 |
Aug 27, 1999 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
427/2.11; 435/287.2; 435/6.1 |
Current CPC
Class: |
B01J 2219/00317
20130101; B01L 3/502715 20130101; B01L 2300/0654 20130101; B01J
2219/00432 20130101; B01J 2219/00511 20130101; B01J 2219/00605
20130101; B01J 2219/00702 20130101; C40B 40/10 20130101; B01J
19/0046 20130101; B01J 2219/00608 20130101; B01J 2219/00711
20130101; C12P 19/34 20130101; B01J 2219/00603 20130101; B01J
2219/00725 20130101; G03F 7/70216 20130101; B01J 2219/0061
20130101; B01J 2219/00621 20130101; B01J 2219/00637 20130101; B01L
3/5085 20130101; Y10S 435/973 20130101; B01J 19/0093 20130101; B01J
2219/00448 20130101; B01J 2219/00704 20130101; C40B 50/14 20130101;
B01J 2219/00479 20130101; G01N 33/551 20130101; B01J 2219/00689
20130101; B01J 2219/00659 20130101; B01J 2219/00436 20130101; C12N
15/10 20130101; G01N 21/253 20130101; B01L 2300/0864 20130101; B01J
2219/00439 20130101; B01J 2219/0059 20130101; B01J 2219/00529
20130101; B01L 2300/0816 20130101; C40B 40/06 20130101; B01J
2219/00441 20130101; B01J 2219/00612 20130101; B01J 2219/00497
20130101; G01N 21/6454 20130101; B01J 2219/00648 20130101; B01J
2219/00722 20130101; B01J 2219/00585 20130101; Y10S 435/97
20130101; B82Y 30/00 20130101; B01J 2219/00596 20130101; B01L
2300/069 20130101; B01J 2219/00675 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 427/002.11 |
International
Class: |
C12Q 001/68; C12M
001/34; B05D 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 28, 1998 |
DE |
198 39 254.0 |
Aug 28, 1998 |
DE |
198 39 255.9 |
Aug 28, 1998 |
DE |
198 39 256.7 |
Feb 19, 1999 |
DE |
199 07 080.6 |
May 27, 1999 |
DE |
199 24 327.1 |
Claims
1. A method for producing a support for determining analytes,
comprising the steps of (a) providing a support body comprising at
least one channel, (b) passing liquid with building blocks for
synthesizing polymeric receptors through the channel or channels of
the support body, (c) site- or/and time-specifically immobilizing
the receptor building blocks in each case on predetermined
positions in the channel or channels and (d) repeating steps (b)
and (c) until the required receptors have been synthesized in each
case on the predetermined positions.
2. The method as claimed in claim 1, characterized in that a
support which comprises defined area regions with, in each case,
identical receptor species is produced.
3. The method as claimed in claim 1 or 2, characterized in that the
channels are arranged on at least one support surface.
4. The method as claimed in any of claims 1 to 3, characterized in
that the support comprises a large number of channels which are
preferably arranged parallel to one another.
5. The method as claimed in any of claims 1 to 4, characterized in
that the receptors are selected from nucleic acids and nucleic acid
analogs.
6. The method as claimed in claim 5, characterized in that the
receptor building blocks are selected from nucleotides,
oligonucleotides, nucleotide analogs and oligonucleotide
analogs.
7. The method as claimed in any of claims 1 to 4, characterized in
that the receptors are selected from polypeptides.
8. The method as claimed in claim 7, characterized in that the
receptor building blocks are selected from amino acids and
peptides.
9. The method as claimed in any of claims 1 to 7, characterized in
that the site- or/and time-specific immobilization of the receptor
building blocks takes place by illumination.
10. The method as claimed in claim 9, characterized in that the
illumination takes place via a programmable light source
matrix.
11. The method as claimed in any of claims 1 to 8, characterized in
that the site- or/and time-specific immobilization of the receptor
building blocks takes place by wetting with an activating fluid
with controllable selection of the activated positions.
12. The method as claimed in any of claims 1 to 11, characterized
in that the pattern of polymeric receptors is fixed by computer
programming.
13. The method as claimed in any of claims 1 to 12, characterized
in that the support is used for determining analytes in a
sample.
14. A method for integrated synthesis and analyte determination on
a support, comprising the steps of: (a) providing a support body,
(b) passing a liquid with, present therein, receptors or building
blocks for synthesizing polymeric receptors over the support, (c)
site- or/and time-specifically immobilizing the receptors or
receptor building blocks in each case on predetermined positions on
the support, (d) where appropriate repeating steps (b) and (c)
until the required receptors have been synthesized in each case on
the predetermined positions on the support, (e) bringing the
support into contact with a sample containing analytes and (f)
determining the analytes via their binding to the receptors
immobilized on the support.
15. The method as claimed in claim 14, characterized in that the
synthesis and analyte determination is carried out in an integrated
apparatus, with the synthesis or/and the analyte determination
process being monitored and controlled in any number of positions
on the support.
16. The method as claimed in claim 15, characterized in that an
integrated apparatus comprising a programmable light source matrix,
a detector matrix, a support arranged between light source matrix
and detector matrix, and means for supplying fluids into the
support and for discharging fluids from the support is used.
17. The method as claimed in any of claims 14 to 16, characterized
in that the analyte is removed again from the support after the
determination.
18. The method as claimed in any of claims 14 to 17, characterized
in that a plurality of synthesis/analyte determination cycles is
carried out, with the receptors or a subsequent cycle being
synthesized on the basis of the information from a preceding
cycle.
19. The method as claimed in claim 18, characterized in that an
extension of the receptors from the preceding cycle takes place for
the subsequent cycle.
20. The method as claimed in claim 18, characterized in that a new
support with receptors which are modified compared with the
preceding cycle is synthesized for the subsequent cycle.
21. The method as claimed in claim 20, characterized in that the
modification of the receptors comprises a change in the sequence
or/and an exclusion of negative receptors from the preceding
cycle.
22. The method as claimed in any of claims 14 to 21, characterized
in that a planar support is used.
23. The method as claimed in any of claims 14 to 21, characterized
in that a support with a large number of channels is used.
24. The method as claimed in any of claims 14 to 23, characterized
in that a plurality of supports is used for a synthesis/analyte
determination cycle.
25. The method as claimed in claim 24, characterized in that the
plurality of supports is synthesized and analyzed in different
detection apparatuses between which there are information
technology links but which may be spatially separate from one
another.
26. A support for determining analytes comprising a large number of
channels, in particular capillary channels, a large number of
different receptors being immobilized in the channels.
27. A support as claimed in claim 26, characterized in that it is
optically transparent at least in the region of the reaction
regions.
28. A reagent kit comprising a support as claimed in claim 26 or 27
and building blocks for synthesizing polymeric receptors on the
support.
29. An apparatus for integrated synthesis and analyte determination
on a support comprising a programmable light source matrix, a
detector matrix, a support arranged between light source matrix and
detector matrix, and means for supplying fluids into the support
and for discharging fluids from the support.
30. An apparatus as claimed in claim 29 additionally comprising
means for deprotection of reaction components on the support.
31. An apparatus as claimed in claim 29 or 30 additionally
comprising electronic control means.
32. The use of the method as claimed in any of claims 1 to 25, of
the support as claimed in claim 26 or 27, of the reagent kit as
claimed in claim 28 or of the apparatus as claimed in any of claims
29 to 31 for determining an analyte in a sample.
33. The use as claimed in claim 32 for the sequencing of nucleic
acids.
34. The use as claimed in claim 33 for new sequencing or/and
resequencing of complex genetic material such as, for example,
individual genomes or synthetic nucleic acids.
35. The use as claimed in claim 32 for obtaining diagnostic
information for individual patient management such as, for example,
the individual effect of pharmaceuticals.
36. The use as claimed in claim 32 for analyzing the effect of
pharmacological substances.
37. The use as claimed in claim 32 for setting up and analyzing
substance libraries.
38. The use as claimed in claim 32 for comparing individuals in a
population.
Description
1 AREA OF APPLICATION OF THE INVENTION
[0001] 1.1 Background
[0002] The precise detection of biologically relevant molecules in
defined investigation material is of outstanding importance for
basic research in the biosciences and for medical diagnostics, and
some other disciplines. In this connection, the genetic information
is present in the form of an enormous variety of different nucleic
acid sequences, the DNA. Realization of this information leads via
the production of transcripts of the DNA in RNA usually to the
synthesis of proteins, which in turn are frequently involved in
biochemical reactions.
[0003] The detection of particular nucleic acids and the
determination of the sequence of the four bases in the chain of
nucleotides, which is generally referred to as sequencing, provides
valuable data for research and applied medicine. In medicine, it
has been possible to a greatly increasing extent to develop,
through in vitro diagnosis (IVD) instruments for determining
important parameters of patients, and to provide the treating
physician therewith. Without this instrument, it would be
impossible to diagnose many diseases at a sufficiently early time.
Genetic analysis has become established here as an important new
method, for example for infectious diseases such as HIV and HBV,
genetic predisposition for certain types of cancer or other
diseases, forensic medicine and a large number of other areas of
application. It has been possible with close interlinkage of
fundamental research and clinical research to trace back and
elucidate the molecular causes and (pathological) relationships of
some disease states as far as the level of the genetic information.
This development is, however, still in its infancy, and much more
intensive efforts are needed in particular for conversion into
therapeutic strategies. Overall, the genomic sciences and the
nucleic acid analytical techniques associated therewith have made
enormous contributions both to the understanding of the molecular
bases of life and to explaining very complex disease states and
pathological processes.
[0004] Further development in medical care will be burdened by the
explosion in costs associated with correspondingly elaborate
methods. In this connection it is necessary not only to push for
implementation of the possibilities for diagnostic and therapeutic
benefits, but also to promote integration into a health system
which is capable of bearing the load and can be financed.
[0005] Use of corresponding technologies in research can likewise
take place on a broad scope and in the academic sector only if the
costs associated therewith are reduced.
[0006] 1.2 Need
[0007] The development of the genomic and proteomic sciences and
the deciphering of the hereditary material are still at an early
stage, as is the realization of the diagnostic potential of a
genetic or gene-manipulative analysis. The methods established to
date are usually labor-intensive and relatively inefficient, which
influences the costs and capacity for example for gaining
information. The most important innovation is the development of
so-called oligonucleotide arrays in which a very large number of
relatively short oligonucleotides of defined sequence are coupled
to a solid matrix (usually silicon) and are thus made available for
parallel hybridization of complementary sequences in the material
to be investigated. The elaborate production and the high cost do
not, however, allow marketing as a mass-produced item at
present.
[0008] 1.3 Fields of Application
[0009] It is intended to employ distinctly cost-reduced systems to
make routine use possible for in vitro diagnostics and clinical
diagnostics, for example of infectious diseases (HIV, HBV etc.) and
their subtypes, for oncology (early tumor diagnosis, tumor
classification, for example type and status), and for determination
of a genetic predisposition.
[0010] It is desirable for fundamental biological research,
especially genomics, to encompass a very large number of
measurement points in the investigated system, for example all
expressed genes. This results in an enormous gain in knowledge in
fundamental biological research (developmental biology, stem cell
culture, tissue engineering, transplantation medicine,
regeneration), which will also lead to important break-throughs in
biomedicine and corresponding applications.
[0011] As has been shown for the. use of DNA chips (Science 280:
1077-1082), it is possible to make a distinction between point
mutations in the base sequence through corresponding biochemical
conditions of hybridization. The system described therein thus
makes extensive screening possible, which can be employed for
forensic purposes, for example for convicting those guilty of
crimes or for detecting family relationships.
[0012] This invention also enables the fast and cost-effective
analysis of foodstuffs, for example for the presence of particular
genes from pathogenic organisms or from genetically manipulated
organisms.
[0013] The screening of medicinal products is likewise of great
importance. The production of, for example, blood products is still
associated with large expenditure ins respect of the safety
measures needed for purity. Screening which is efficient in terms
of both time and costs for such samples is made possible by this
invention in order, for example, to prevent contamination with
infectious material (HIV, HBV, HCV etc.).
2. PRIOR ART
[0014] Biochips are miniaturized hybrid functional elements with
biological and technical components, for example biomaterials which
are immobilized on the surface of a support and which can act as
specific interaction partners (for example DNA oligonucleotides),
and a silicon matrix. These functional elements are usually
arranged in rows and columns, in which case they are called biochip
arrays. Since thousands of biochemical functional elements can be
arranged on the biochip, they must be produced by microengineering
methods.
[0015] Enormous funds are being used, especially in the USA, to
promote the development of miniaturized biochips. The most
important companies active in this field are listed below:
[0016] Affymetrix, Beckman Instruments, Blue Chip Biosystems,
Caliper Technologies, Cura-Gen, Genometrix, Gene Trace Systems,
Hyseq, Incyte Pharmaceuticals, Molecular Tool, Nanogen, Pharmacia,
Synteni, Third Wave Technologies, Vysis.
[0017] Biochips disclosed to date can be classified by the
following criteria:
[0018] Detection principle:
[0019] Chromatographic methods
[0020] Interaction of analytes with solid phase, usually
immobilized interaction partners (for example hybridization of
nucleic acids on DNA oligonucleotides).
[0021] Detection methods (optical, electrical).
[0022] Marker-based detection methods (for example absorption,
fluorescence or luminescence) or marker-free detection methods
(generation of light to detect the reaction).
[0023] Assignment of the analyte to its support [solid phase]
(array, with more than one immobilized interaction partner per
support or single, with only one immobilized interaction partner
per support).
[0024] Production method (for example photoactivated synthesis of
oligonucleotides directly on the biochip, spot completely
synthesized oligonucleotides, coat beads or tubes).
[0025] Types of support (glass chips, plastic chips, microtiter
plates, tubes or beads).
[0026] Presentation for detection (serially, in parallel).
[0027] Optical detection (serially in a scanner or in parallel with
a CCD camera).
[0028] Among the firms listed, only Affymetrix uses the principle
of photolithography for generation of high density DNA arrays on a
planar surface, whereby it has made by far the greatest advances in
the parallelization of oligo sequences detection.
[0029] GeneChip from Affymetrix Inc., Santa Clara, Calif.:
[0030] Production takes place by in situ synthesis of DNA
oligonucleotides on planar chips in high density (July 98: up to 64
000 different oligos on 1 cm.sup.2). The production method is based
on photolithography, which is used and has been optimized in the
semiconductor industry and which entails use of photoactivatable
binding of oligos to the chip surface as well as to oligos already
present. Production takes several hours due to the large number of
process steps. Detection takes place by serial optical detection of
the planar chip in a fluorescence scanner. Hybridization of the
sample on a chip takes about 1.5 hours. First products (sequencing
chip for tumor marker p53 exons 2-11, breast cancer gene BRCA1 exon
11, HIV GeneChip) are already commercially available. The costs at
present are in the region of several hundred dollars for one
GeneChip, additionally a detection unit is also required.
[0031] Further relevant prior art are WO91/18276, EP-A-0 671 626
and EP-A-0 430 248.
3. SUMMARY OF THE INVENTION AND OBJECT ACHIEVED THEREBY
[0032] The invention relates to a method as claimed in claim 1 for
producing a support for determining analytes, comprising the steps
of
[0033] (a) providing a support body comprising at least one
channel,
[0034] (b) passing liquid with building blocks for synthesizing
polymeric receptors through the channel or channels of the support
body,
[0035] (c) site- or/and time-specifically immobilizing the receptor
building blocks in each case on predetermined positions or regions
in the channel or channels and
[0036] (d) repeating steps (b) and (c) until the required receptors
have been synthesized in each case on the predetermined positions
or regions.
[0037] Claims 2 to 13 relate to preferred refinements of this
method. The support is a solid phase which can be or is charged
with biologically or chemically functional materials or receptors
(probes) or building blocks thereof. In this embodiment of the
invention, the support has a surface which is provided with
depressions, for example at least one channel and particularly
preferably with a large number of channels. The channels are
preferably microchannels with a cross section of, for example, 10
to 1 000 .mu.m. The channels may be--depending on the surface
properties--capillary channels, but also channels without capillary
action (for example because of Teflon coating). The support is
preferably optically transparent at least partly in the region of
the positions or regions to be charged with receptors. The regions
of the support which are to be charged with receptors are
preferably chemically and physically identical to one another, i.e.
they have essentially identical surface characteristics.
[0038] The invention further relates to a method as claimed in
claim 14 for integrated synthesis and analyte determination on a
support, comprising the steps of
[0039] (a) providing a support body,
[0040] (b) passing a liquid with, present therein, receptors or
building blocks for synthesizing polymeric receptors over the
support,
[0041] (c) site- or/and time-specifically immobilizing the
receptors or receptor building blocks in each case on predetermined
positions or regions on the support,
[0042] (d) where appropriate, repeating steps (b) and (c) until the
required receptors have been synthesized in each case on the
predetermined positions or regions on the support,
[0043] (e) bringing the support into contact with a sample
containing analytes to be determined and
[0044] (f) determining the analytes via their binding to the
receptors immobilized on the support.
[0045] Claims 15 to 25 relate to preferred refinements of this
method. It is also possible to use planar supports in this
embodiment.
[0046] Claim 26 relates to a support for determining analytes
comprising at least one channel and preferably a large number of
channels, in particular capillary channels, whereby a large number
of different receptors are immobilized in the channels. The support
is preferably optically transparent at least in the region of the
regions to be charged with receptors.
[0047] The invention further relates to a reagent kit as claimed in
claim 28 comprising a support as described above, and building
blocks for synthesizing polymeric receptors on the support. The
reagent kit may additionally comprise reaction liquids for
synthesizing the receptors on the support.
[0048] The invention also relates to an apparatus for integrated
synthesis and analyte determination on a support as claimed in
claim 29 comprising a programmable light source matrix, a detector
matrix, a support arranged between light source matrix and detector
matrix, and means for supplying fluids into the support and for
discharging fluids from the support. The programmable light source
or illumination matrix may be a reflection matrix, a light valve
matrix, for example an LCD matrix or a self-emitting illumination
matrix. Claims 30 and 31 relate to preferred refinements of these
apparatuses.
[0049] Finally, the invention also relates to the use of the
claimed method, support, reagent kit and the claimed apparatus for
determining an analyte in a sample. Claims 33 to 38 relate to
preferred applications.
[0050] One embodiment of the present invention is represented by a
method and system for cyclic integrated synthesis and analysis,
which is to be referred to as the ISA system. Direct coupling,
which is preferred according to the invention, of synthesis and
analysis makes high throughput determination of analytes, which is
a distinct improvement over the prior art, possible in a cyclic
method. It is possible in its connection for the substances to be
analyzed to be, for example, in the form of segments or fragments
or a larger molecule chain.
[0051] In a preferred embodiment of the invention, a direct logical
linkage is provided between the results of the analysis of a first
support and the synthesis of the support which is to be
subsequently produced, thereby making it possible to transfer the
information gained in a preceding cycle to a subsequent cycle. In
this way there is stepwise development of learning of the
analytical system.
[0052] Said cyclic sequence of synthesis, sequence comparison,
analysis of the comparative results and renewed synthesis of
receptors on the support can be repeated as often as desired--until
a desired termination criterion, which can be chosen as
required--is reached.
[0053] The feedback, and the learning process associated therewith,
from the preceding cycle makes the method of the invention and the
apparatus also suitable for research on very large and complex
analyte molecule chains, for example for sequencing in individual
genomes, such as the human genome. The expenditure of time in this
case is improved compared with the prior art by at least one
hundred-fold, more probably by one thousand-fold and potentially by
10 000-fold.
[0054] The method can be employed for "new sequencing" of unknown
nucleic acid sequences (DNA, cDNA, RNA) including their spatial
arrangement, or mapping. It is possible with this procedure to
produce an individual gene profile of each individual and each
species, whether by sequencing parts of the genome or of the whole
genome.
[0055] The method can additionally be employed for "resequencing"
of nucleic acid sequences, i.e. for comparing previously known
sequences (represented in the form of the receptor probes) with
unknown sequences in the sample to be investigated. The known
sequences are selected appropriately for the problem and
specifically for this purpose.
[0056] The described resequencing allows the user to generate
individual polymeric receptors on site on the support of the
invention starting from a neutral support and subsequently to
immediately carry cut an analysis of the sample to be investigated.
This possibility results in a maximum diversity of variants of the
receptors with a minimal space requirement.
[0057] It is possible by combining new sequencing and resequencing
to adapt diagnostic tests or medicines to the needs of an
individual at short notice.
[0058] It is possible with exceptional flexibility to analyze
expression patterns as a further important area of application. The
corresponding receptors or polymer probes for this purpose are
usually selected on the basis of known sequences. The use of the
method for determining gene expression can also take place in the
context of high throughput screening.
[0059] In addition, different approaches to screening methods and
the setting up and analysis of substance libraries are conceivable
with various naturally occurring and artificial receptor probes.
This may take place, for example, in connection with the search for
and the characterization of pharmacologically active
substances.
[0060] The fields of application of the method of the invention and
the apparatus of the invention for cyclically integrated synthesis
and determination of analytes are wide-ranging and extend in
principle to all analytical applications such as gas
chromatography, thin-layer chromatography, gel electrophoresis,
capillary electrophoresis, mass spectrometry etc. The same applies
in principle to all applications of highly parallel solid-phase
analysis.
[0061] There is no longer any need at all to store complex
polymeric receptors ready for use. In addition, there is no
physical restriction on the number and selection of the receptors.
The required number of receptors can be distributed over a
plurality of reaction supports or a plurality of cycles in a
reaction support, because the individual receptors are subject to
no site specifications for logical evaluation of the comparative
results.
[0062] The present invention relates to a novel "support" as basis
for the use of a preferably light-controlled synthesis of
individual bases (G, A, C and T) or oligonucleotides (base
sequences) to form a highly parallel, planar and dense arrangement
(array) of these oligonucleotides in a solid support matrix
(chip).
[0063] The novel biochip, the "optofluidic microprocessor",
comprises a structure of microchannels, preferably capillaries, in
an at least partially transparent and preferably flat body. On
synthesis or immobilization of receptors, the liquid starting
materials are passed through the channels in the support and bind,
locally activated, to the channel walls. This creates the technical
requirements for a rapid, efficient and thus cost-effective
production, which will make wide use of these supports possible.
The density and parallelity are of the same order of magnitude as
for competing techniques, with several hundred thousand defined
oligonucleotides on a support. The advantage of the novel technique
is the more favorable physicochemical properties of the flow and
wetting processes in the channels compared with a uniform
surface.
[0064] Production of the chips consists of producing a support
body, which is preferably provided with microchannels, from a
suitable, light-transmitting material, and of the biochemical
coating process, preferably on the walls of the individual
microchannels, so that subsequent synthesis of the polymeric
receptors, for example oligonucleotides, in the channels is
possible. This entails site-specific attachment of individual
receptor building blocks, oligomeric synthons (for example di-,
tri-, tetra- or pentanucleotides) or whole base sequences (oligos)
in the individual channels in the support by means of
photoactivation by a suitable light source. This results in a large
number of receptor-charged regions (specific binding or
hybridization sites) in each channel, and each region serves,
because of its individual receptor-sequence combination, for the
binding and subsequent detection of a specific analyte, for example
a DNA fragment. The regions are separated from one another in one
dimension of the planar support by the walls of the channels, and
with photoactivated binding a corresponding free space is left
between two adjacent regions along the individual channels. The
result is a highly parallel, highly integrated array of specific
receptors. Because of the possibility of multiplexing
oligosequences and parallel channels (for details, see section 5) ,
it is possible to reduce the production times to 1/4 on use of
single bases, 1/8 with dinucleotides and to {fraction (1/16)} with
trinucleotides by appropriate multiplexing of the oligos (starting
materials) and of he channels to be wetted. This also makes
flexible adaptation to customers' requirements, the "tailored"
biochip, possible. This systematic speeding up is not possible in
planar systems (planar chips).
[0065] For the analysis, the investigational material (for example
DNA, RNA in solution) is passed through the channels and has the
opportunity to bind to the receptors, for example by hybridization
onto complementary strands, if these are present. It is preferred
to use high-resolution, parallel CCD chips for detection and
evaluation of the particular analyte binding, for example a DNA
hybridization. The binding of the analyte to the immobilized
receptor is [lacuna] by suitable signal-emitting groups known from
the prior art, for example light-emitting groups. However, novel
detection methods can also be applied. For detection it is possible
to do without optically imaging lens systems if the size of the
channels is chosen so that each measurement point covers a
sufficient number of pixel elements of the detector, for example of
a CCD chip. This direct usage (no optical system) of highly
parallel CCD matrix chips with a large number (currently 16 million
pixels per 1 cm.sup.2; research status: 80 million pixels per 1
cm.sup.2) of pixels (optical sensors) makes it possible to detect a
large number of light signals in parallel (see BioScanner from
Genometrix). Therefore it is attempted even for the detection unit
to have recourse to a high-tech product fabricated in large numbers
and at low cost in place of costly optical arrangements.
[0066] The invention thus covers the essential requirements for DNA
analysis, namely simultaneous determination of a large number of
DNA sequences (achieved by highly integrated, miniaturized supports
and high-resolution optical detection), provision of cost-effective
tests (multiplexing in production, low-cost disposable supports,
for example injection-molded, rapid synthesis during production),
rapid procedure for the analysis due to small volumes and favorable
wetting processes, reduction in starting materials through the flow
geometry of the support etc., rapid evaluation (achieved by
parallel optical evaluation in planar arrangements [DNA chip
array]), a cost-effective analytical system (achieved by dispensing
with costly, microsystem and optical components) and ensuring
quality both during production and during analysis (achieved by
defined flow processes in the support).
[0067] The use of photoactivation of chemical reactions in the area
of the support synthesis leads, in particular in combination with
the technology platform of the opto-fluidic microprocessor together
with a programmable light source matrix, to the breakthrough,
because this makes it possible to reduce the production costs for a
single support while, at the same time, improving the quality, by a
factor of 10-100. In this way, a cost-effective, massively
parallel, highly integrated and, at the same time, easily
miniaturizable and automatable DNA chip technology is made
available for the first time.
[0068] Despite the complex data evaluation, only a minimum of
different hardware components is required because the support
bodies which need to be changed either for each cycle or only when
worn are initially all--before the start of the receptor
synthesis--identical. All individuality results only from the
specific receptor synthesis and from the information obtained
stepwise by the analysis which, after the synthesis/analysis cycle,
is converted back into information, so that the individuality, i.e.
the characterizing features of the biological/chemical material,
are once again present only in the form of electronic data.
4. MAIN FEATURES OF THE MODE OF ACHIEVEMENT
[0069] The mode of achievement in principle in this system is based
on stepwise biochemical synthesis of receptors on the surfaces of a
large number of cannel walls on a support. These channels are
arranged on the support, for example a small planar chip. The
synthesis takes place with the appropriate bases or multi-base
oligonucleotides (base sequences) by photoactivated site-specific
binding. The wetting of these specifically "labeled" channels with
the DNA analytes to be investigated and the subsequent detection of
the binding reaction via suitable signal-emitting groups concludes
a cycle of the method.
[0070] 4.1 Microstructure as Support Matrix
[0071] The support synthesis comprises the provision of the support
body, which preferably consists of a suitable, light-transmitting
material, and the biochemical generation of receptors on the walls
of the individual channels. The specific synthesis of the receptors
can take place either directly during production of the support
body or not until used.
[0072] Various materials (for example glass, silicon, ceramic,
metal or plastic) can be used for the support bodies. It is
important that the walls of the channels satisfactorily transmit
both the excitation waves for the photoactivated synthesis and the
light waves (where appropriate excitation and reaction signal) for
the subsequent detection (analysis). Depending on which material is
employed, the walls of the channels must be coated with a reactive
material so hat the receptors or receptor building blocks can bind
to the surface.
[0073] The geometry of the supports corresponds, for example, to a
"check card", and the size of the area covered by the channels is
determined by the CCD chip used for detection. Various methods can
be employed to produce the channels in the support. Account must be
taken on the influence of the cross-sectional geometry of the
channels, which has a great influence on the resulting hydrodynamic
forces and the possibility of cleaning the channels. Methods which
can be used for production are, for example, laser, milling,
etching techniques or injection molding.
[0074] The following aspects must be taken into account in the
arrangement of the channels in the plane: if a large number of
parallel channels is used, it is possible to minimize the synthesis
times, but the wetting or filling of the individual channel is
correspondingly complex. If, at the other extreme, there is only a
single long channel, the synthesis is correspondingly slow because
the multiplexing of channels to bases or whole oligos cannot be
used, and all processes can take place only serially one after the
other. The advantage of only one channel is for the analysis, where
the sample flows past each measurement point in all the
channels.
[0075] 4.2 Synthesis Cycle in the Support
[0076] The positions (reaction regions) intended for coating with
receptors in a support body are filled with one or more fluids
through channels from containers via feed lines, valves and
fittings. It is possible with the aid of a light emission/detection
unit which is disclosed in German patent application 198 39 254.0
and which is preferably a programmable light source or illumination
matrix, as described in German patent application 199 07 080.6, to
illuminate selected positions or regions on the support and, in
this way, control the individual synthesis of receptors, the
support being in this connection an optofluidic microprocessor. In
place of illumination, the selected reaction regions can also
undergo individual fluidic activation. After completion of the
reaction, the reaction regions are rinsed and refilled, after which
another activation cycle follows.
[0077] The progress of receptor synthesis can be followed and
controlled by means of suitable detection units.
[0078] As soon as the synthesis of the receptors is completed, the
reaction regions are cleaned and are then available for an analyte
determination method.
[0079] 4.3 Nucleic Acid Analysis Using Oligochips--Basic
Principle
[0080] As already shown for several arrangements (for example
Molecular Medicine Today, September 97, pp. 384-389; Trends in
Biotechnology, November 97, pp. 465-468), it is possible to use the
hybridization of nucleic acid strands onto a, usually short,
complementary sequence, a so-called oligonucleotide or oligo, for
sequence analysis. For this purpose, high-density arrangements of
synthetic oligonucleotides are generated onto a solid matrix and
permit multiple parallel hybridization experiments. The leading
method (August 98) is a photolithographic and thus local activation
of synthesis precursors. Based on the technique which has been
learned from the production of microelectronics, the parallel
arrangements are referred to as chips.
[0081] An enormous analytical capacity is produced by a massive
increase in the number of reaction regions ("measurement points"),
i.e. defined oligos at a defined site.
[0082] The sample to be investigated normally contains DNA or RNA.
It may be necessary to isolate and replicate these in an
amplification step (for example PCR), and moreover acquire a label,
for example a dye, fluorescent or luminescent label.
[0083] Sequencing of a DNA molecule is also possible through a
sufficiently large number of receptor-charged regions (reaction
regions) (Sequencing-by-Hybridization SBH, see BioTec March 98, pp.
52-58), and other applications show the determination of point
mutation polymorphisms (i.e. differences between individuals in
single bases in a defined DNA section) and permit, inter alia,
identification of such polymorphisms in hundreds of subjects in
parallel (Science 280, May 98, pp. 1077-1082).
[0084] The investigation of whole genomes and of the gene
expression status of whole cells also becomes possible for the
first time (for example Proc. Nat. Acad. Sci. USA 95, March 98, pp.
3752-3757).
[0085] The invention described herein accordingly allows the use of
a large number of established methods for investigating nucleic
acids and genetic material. This is simultaneously associated with
a large increase in such applications and thus an enormous economic
advance, because it is expected that the optofluidic microprocessor
will provide such technology more flexibly than available methods
and at distinctly lower costs.
[0086] 4.4 Photoactivated Synthesis of Oligonucleotides and
Peptides on the Support
[0087] In the assembly of receptors on the support there is
site-specific addition of receptor building blocks, for example
single bases (G, A, C, T) or oligonucleotide sequences (preferably
about 2 to 4 bases long) in the individual regions by means of
photoactivation by a suitable light source. The channels are
sequentially filled with the synthesis building blocks, for example
G, A, C and T, and irradiated site-specifically along the channels
with high-resolution light of a particular wavelength and
intensity. Between the coating cycles, the channels are
appropriately rinsed in order to remove unbound receptor building
blocks.
[0088] This results in a large number of reaction regions (specific
binding or hybridization sites) in each channel, each reaction
region serving, because of its individual receptor sequence, for
the binding and subsequent detection of a specific analyte, for
example a DNA fragment. The reaction regions are separated from one
another in one dimension of the planar support by the walls of the
channels, and in the second dimension, along the individual
channels, a corresponding free space is left between two adjacent
reaction regions on photoactivation.
[0089] Photolithography can also be used for the photo-activated
binding of the receptor building blocks. However, other methods can
also be employed.
[0090] An illumination method using a programmable light source
matrix, for example a self-luminous light source matrix, a light
valve matrix or a reflection matrix, whose matrix points or light
source elements can be deliberately controlled, in particular in
relation to the intensity and, where appropriate, color of the
light, is particularly preferably carried out. Thus, it is possible
with such a matrix to generate in each case the required
two-dimensional illumination patterns in a simple manner, in
particular in a computer-assisted manner. The preferred
photoactivation of the oligos for producing the support is effected
directly by the illumination matrix. The wavelength necessary for
this, for example 365 nm (upper UV region near to visible light),
can be controlled with all variants of the programmable light
source matrix.
[0091] It is also possible to assemble receptors from amino acid
or/and peptide building blocks in a corresponding way.
[0092] 4.5 CCD Chip Detection of the Specific Detection
Reaction
[0093] As described, the binding of a DNA analyte is to lead
directly or indirectly to a detectable signal, for example a light
signal. This can take place, for example, by absorption, an
exciting light (fluorescence) or by photon emission (luminescence).
The signal is detected preferably by use of a CCD chip which is
preferably placed directly underneath the support. The excitation
light source is preferably placed over the support and,
correspondingly, the translumination method is used for
measurement. Each light signal can be detected on the CCD chip, in
particular differentially according to intensity and, if required,
also according to wavelength (color). The recorded spectrum can be
evaluated qualitatively or quantitatively. In addition,
differentiation of wavelengths and intensities also allow signal
sources to be differentiated.
[0094] The types of excitation light for the detection method must
be chosen to be monochromatic (for example laser light for
fluorescence excitation) or heterogeneous (for example white light
for absorption measurement) depending on requirements.
5. IMPROVEMENTS AND ADVANTAGES COMPARED WITH CURRENT SYSTEMS
[0095] The novel supports overcome the disadvantages, listed below,
of mask-based photolithography methods or in situ spotting.
[0096] The principle of extended wetting of the entire chip surface
with fluid does not permit any multiplexing in production. Thus,
the number of production cycles for 20 base-long oligos increases
on use of dinucleotides (4.sup.2=16 possibilities) from
4.times.20=80 hybridization steps to 16.times.10=160, which means a
doubling. The same also of course applies to the intermediate
washing cycles.
[0097] Synthesis of the photoactivatable bases on the planar chip
surface, just like the required washing steps in the production of
chips, cannot be achieved except by dipping processes (chip is
dipped in the liquid) which involve much space and manipulation, or
rinsing processes along the surface, which involves much liquid,
(for example centrifugation principle from semiconductor
technology), which represents a very great impediment of
miniaturization and automation from the viewpoint of equipment
development.
[0098] In the subsequent DNA sequence detection, uniform
distribution of the sample on the chip surface is complicated (no
simple and thus reliable mixing method is possible) and a
correspondingly large amount of sample fluid is necessary. The
search for a rare event in the sample is impossible because
adequate contact of all constituents of sample with all specific
measurement points cannot be ensured.
[0099] 5.1 Reduction in Production Times Through Multiplexing in
the Synthesis
[0100] The essential advance of the novel supports is the
possibility of drastically reducing the production times for the
individual synthesis of the receptor-charged supports through
appropriate multiplexing between receptor building blocks as
starting materials and the channels.
[0101] For site-specific generation of a large number of different
receptor sequences, for example base sequences of a particular
length (for example 20 bases) on a planar surface by means of
locally high-resolution photoactivation, 4 (owing to the 4
different bases) synthesis cycles are required in each plane
(calculation example: 20 bases in each base sequence) of the DNA
chip array. There are accordingly 4.times.20=80 cycles for 20 base
planes. On use of dinucleotides (2 bases) on the same surface, 2
planes are produced all at once, but 4.sup.2=16 synthesis cycles
are necessary for these 2 planes. Accordingly, 10.times.16=160
synthesis cycles are required for 20 plates, instead of 80 cycles,
which means a doubling of the production times. On use of
trinucleotides (3 bases), this effect is amplified to more than
five times the number of cycles. Thus, with a single planar
surface, the use of individual bases is the fastest possibility for
photoactivated DNA chip production. There is no possibility of
reducing the number of synthesis cycles.
[0102] The synthesis of the optofluidic support differs from this
in that there is the possibility of distributing the starting
materials, i.e. the bases or the different variants of
dinucleotides (4.sup.2=16 combinations) or trinucleotides
(4.sup.3=64 combinations) to various channels. This means that, at
least in the lower planes near the support, only one base or one of
the possible base sequences is always introduced into each channel.
Depending on the specified total number of base sequences to be
generated in the channels of the support, it may be that this
principle must in some cases be set aside in the upper planes, i.e.
more than one base or oligo must flow through one of the channels
for one base, dinucleotide or trinucleotide plane. Once again, this
increases the number of synthesis cycles somewhat where
appropriate. However, overall, there is still a very large
reduction in the production times to theoretically 1/4 of the
cycles with single bases, 1/8 of the cycles with dinucleotides and
to {fraction (1/16)} of the cycles on use of trinucleotides as
starting materials for receptor synthesis (and so on for longer
oligos). The number of cycles required for a specific support is
individual for each support and can be stated only as a statistical
average when the number of reaction regions on and in the support,
the number of parallel channels and the length of the oligos to be
synthesized on the support is predetermined. Optimization of the
synthesis times or a support is to take place by means of a
software tool to be developed (for example CAMS Computer Aided
Multiplexing Synthesis) which is integrated in the control of the
analytical system to be developed or in the interfaced
computer.
[0103] 5.2 Reduction of the Starting Materials and Quality
Assurance
[0104] The use of channels very greatly reduces the amount of fluid
required and, at the same time, increases the quality both in the
synthesis of the support and in the subsequent detection of a
sample compared with the use of a single area. Thus, the uniform
wetting of channels is hydrodynamically very simple, consumes
little fluid and therefore can be miniaturized and automated very
easily. This applies in particular also to the need for adequate
quality of the channel washing processes.
[0105] The fluid required is already reduced by 50% by the walls of
the channels which, in principle, cover the space between two
reaction regions in the support array. This applies both to the
coating of the support during production, the synthesis of the
receptors and to the "sample loading" for the analysis. A further
reduction in the amounts of fluid results from the good wetting of
the channel walls by a fluid flowing through and, in particular, by
the effective washing processes which can, for example, be greatly
improved by "cleansing" gas bubbles in the channels. On the other
hand, good, statistically adequate distribution of the sample on a
surface can be achieved only with a very large amount of
sample.
[0106] A further advantage of the channels is that the cycle times
are shorter, resulting from the smaller volumes of fluid and,
associated with this, the faster chemical reactions and operations.
This results in both synthesis and hybridization times being
shorter.
[0107] This additionally results in a distinct reduction in errors
both in production and in detection, which further increases the
number of measurements which can be evaluated per usage of material
and time, and forms the basis for quality assurance based on
accurately definable and reproducible flow processes.
[0108] The simple miniaturization and automation of the operations
in the novel supports form the basis for simple miniaturization and
automation of the entire novel analytical system based on the
supports.
[0109] 5.3 Three-Dimensional Reaction Surfaces
[0110] It is possible by suitable design of the cross-sectional
geometry of the individual channels to increase the useful reaction
surface. The size of this area is just as important for the
addition of the oligos during production as for the accumulation of
the DNA fragments from the sample which are flowing by, and the
intensity of the light signals resulting from hybridization.
[0111] Thus, a rectangular channel has, provided the height and
width are identical, on use of the walls and the top surface four
times the reaction surface for an identical base area, i.e. the
same space requirement in the two dimensions of a planar support.
Even if hydrodynamic requirements lead to the channels having a
round design inside (for example possibilities of better cleaning
by gas bubbles in the channel), the reaction surface is still about
three times that with a planar surface. The use of this
three-dimensional flow geometry makes possible to reduce further
the starting material requirement (production and analysis).
[0112] Another effect can likewise be influenced by the
cross-sectional geometry of the channels: the reflection of light
at the transition from the interior of the channels to the
surrounding medium of the support. Thus, any curvature has either a
focusing or scattering effect on the direction of propagation of
the light. Thus, the light paths can be optimized in the support by
appropriate choice of the upper and lower sides of the flow channel
geometry.
[0113] 5.4 Parallel CCD Chip Detection
[0114] Measurement of the light signals of all the reaction regions
of the support "all at once" makes use of the continually growing
potential of the high-resolution CCD camera chips. These allow
detection of all light signals for reaction or hybridization
detection in a single measurement procedure. For this purpose,
current color CCD chips provide about 3 000.times.3 000 pixels with
a pixel size of about 10.times.10 .mu.m on an area of 40.times.40
mm. The state of research is already at corresponding CCD chips
with about 4 000.times.6 000 pixels. Signal detection takes place
synchronously for all pixels in fractions of a second. This means
that there is a great growth potential also for the described
application of CCD chip technology, and parallel detection of
10.sup.6 individual reaction regions in the support is technically
feasible. This avoids the time-consuming scanning procedures of
conventional systems, and the pure measurement time is reduced to a
minimum and becomes entirely insignificant in relation to other
steps in the method.
[0115] Processing of the resulting quantities of data is possible
without difficulty owing to the development in efficiency with a
simultaneous fall in price of modern computer systems.
[0116] 5.5 Direct Detection without Optical System
[0117] Direct detection of the light signals, without an optical
system, by a CCD chip has the advantage of a considerably smaller
amount of energy required by the light for error-free detection.
Such an arrangement is said--investigated in a different
connection--to consume only 10% of the amount of excitation light
of a comparable arrangement with an optical system. In other words,
the optical system consumes 90% of the light energy. The lower
intensity of light greatly reduces unwanted light-scattering
effects in the support surrounding the channels, as well as the
possible need to cool the light source used. In addition, omission
of an optical system means a great saving in space and a reduction
in the production costs for the detection unit.
[0118] Complicated units for moving the support or the detection
unit, as are necessary in scanners, are likewise entirely dispensed
with. The predetermined dimensions of the CCD chips (several
cm.sup.2) make it possible to use a very large number of parallel
channels (several 100) with a moderate channel size (in the 10-100
.mu.m range).
[0119] 5.6 Disposable Supports
[0120] The supports can be designed as simple disposables
(disposable chips). Possible in principle are either glass,
silicon, metal, ceramic or plastic chips (cost-effective injection
molding methods) and other embodiments.
[0121] The biochips of other technologies are likewise designed as
disposables for a few measurements. However, in this case, the very
high cost owing to the complicated production of the chips is
usually not in favor of disposing of the chip after only one or a
few measurements.
[0122] 5.7 Flexibility of Use
[0123] The rapid and cost-effective production makes a wide variety
of individual applications possible, in which, for example,
oligonucleotide arrays are specifically synthesized taking account
of sequence and gene databases on the Internet.
[0124] It would be possible by use of a single, multiply coiled or
spiral channel to establish a hybridization in the (slow)
flow-through, which also makes it possible to detect rare events
(for example rarely expressed genes). This would introduce a
chromatographic principle into DNA array technology.
[0125] The use of di-, tri- or longer oligonucleotides as synthetic
building blocks makes it possible to achieve a further reduction in
the production times. It is possible, especially for simpler
arrays, for synthesis units to be used directly by the customer,
and thus for the composition of the array to be definitively
individualized.
[0126] The great flexibility of the technology is also important in
view of the finding that the genes of single individuals vary
greatly so that it is not possible to construct a general gene
catalog for all species. The support makes it possible in this
case, for example in a first measurement cycle, to match the basic
data which are provided on the Internet--freely accessible or only
specifically for the customers of the system--with the individual
differences of a patient and, from the results, to form a
corresponding second DNA array which carries out the actual tests
adapted for the individual.
[0127] The achievement according to the invention can also be used
to synthesize peptide sequences in the channels. This would provide
highly complex and, at the same time, cost-effective peptide arrays
for a large number of applications.
6. Review of Some Aspects of the Invention
[0128] 6.1 Support Design Variants
[0129] There is a large number of design variants both for the
configuration and for the fabrication of the supports. In the
arrangement of the channels in the support over the area of the
detection unit it is just as conceivable to use only one channel as
to arrange a large number of parallel channels. Thus, there are no
technical difficulties in fabricating on an area of 25.times.37 mm
an arrangement of 500 channels (state of the art: 500 parallel
capillaries with a diameter of 900 nm) with a length of 37 mm and
in each case about 750 reaction regions. The same number of
reaction regions (500.times.750=375 000) could also be accommodated
in a single serpentine channel with a length of about 20 m.
[0130] The advantage of only one channel is that the sample is
presented at all measurement points of the array and is therefore
particularly suitable for searching for rare constituents. A large
number of parallel channels has the advantage that the production
times for the support synthesis can be minimized through the
multiplexing of starting materials and channels and all the flow
processes. This channel arrangement is therefore to be preferred
for support synthesis and all analyses with a sufficient number of
copies of each analyte in the sample.
[0131] In order to benefit from both advantages in one support, it
is possible to introduce the starting materials for the support
synthesis by means of parallel fittings at the entry to the
channels, although the channel consists of only a single, long
microchannel from the sample input onward. This effect can also
take place through the integration of valves in the support or the
surrounding equipment components. Thus, Biacore has designed valves
controlled by fluid in a two-part injection molded chip by a
membrane which presses from below into the channels on the upper
side of the chip and thus closes the channels.
[0132] A large number of structures and microchannel courses is
possible as arrangement of the channels on the detector area.
Parallel or "snake-shaped" structures, for example, are obvious for
high parallelity of the fluidic processes. The division of the
channels should in this case take place according to the duality
principle, where two new channels arise from each channel, and all
of them are of equal length. Thus, 10 divisions result in
2.sup.10=2 048 channels. Spiral arrangements have the advantage
that their flow processes are less turbulent and their cleaning is
better. Their great disadvantage is the feeding in and out, which
must take place in the third dimension upward or downward, which is
rather unfavorable in terms of fabrication techniques and
optically.
[0133] A possible material for the support is, for example, glass,
silicon, ceramic or metal or/and plastic. A two-layer structure is
possible, and the layers can be joined together for example by
gluing or bonding, or not. The structure of the channels may in
this case be introduced either only into one or else into both
sides or halves. Fabrication methods which can be used for this
purpose are, inter alia, laser or precision milling. Injection
molding is particularly cost-effective and allows adequate quality
of fabrication. Other methods are the LIGA technique or hot
molding.
[0134] 6.2 Support Synthesis
[0135] There are in principle two possibilities for synthesizing
the individual capture receptors, for example oligos, on the
reaction regions in the support array. The customer purchases
finished supports from the manufacturer with a predetermined
selection of immobilized base sequences, or he synthesizes his own
chosen sequences on unlabeled supports in a synthesis unit.
Information about appropriate sequences can be found, for example,
in databases on the Internet, where they are provided freely or
else specifically by the support manufacturer.
[0136] 6.2.1 Synthesis Unit
[0137] The synthesis unit consists of a suitable light source which
irradiates site-specifically, with great accuracy and exact
resolution, the reaction regions in the support array on synthesis
of the receptors, for example bases or base sequences, on the
support surface or the channel walls. As already mentioned under
4.4, the illumination can take place by means of a programmable
light source matrix. It is also possible to use a photolithography
unit like those employed in semiconductor chip production for the
photoactivated etching of Si wafers.
[0138] 6.2.2 Finished Support Synthesis by the Manufacturer
[0139] In the case of marketing of finished supports, the
manufacturer carries out the synthesis. The latter requires for
this purpose an appropriately efficient synthesis unit which uses
oligos which are as long as possible (3 or more bases long) as
starting materials, which are introduced (injected) in parallel
into the channels, and thus minimize the synthesis times for each
support (multiplexing). It is possible in this case to provide
special accesses in the supports with the aim of obtaining the
maximum number of parallel and thus short channels, irrespective of
the channel structure provided for the analytical procedure.
[0140] 6.2.3 Starting Materials in the Support
[0141] For applications where individual configuration of the
arrays, but not rapid synthesis of the supports, is what matters,
it is possible to provide the starting materials (G, A, C, T and
buffer etc.) directly in the support in corresponding reservoirs.
The excess starting materials must be collected in a corresponding
chamber in the support. The volume of such a chamber can be
designed without difficulty to have a multiple of the total channel
volume through an expansion in the third dimension upward or
downward. One conceivable application of this support variant is
particularly for research laboratories, but also for small medical
practices.
[0142] The principle of capillary force can in this connection be
used in a possible design variant directly for fluid transport in
the support. No mechanical system would be used, and the filling of
the capillaries with the starting materials and the sample could
take place by simply adjusting a valve in the support. The "waste
chamber" could display a supporting suction effect through
embedding a suitable nonwoven material. In order to minimize the
required amounts of fluid, care should be taken that the
capillaries are always of the same length in these one-way flow
designs (no circulation and thus no reuse of the starting
materials). This is likewise important for the functioning of the
capillary force as pump.
[0143] A further variant is vertical alignment of the planar
supports, so that gravitational forces can also be utilized for
fluid transport in the support. If these forces are insufficient to
achieve all the necessary fluid transports into the support, other
suitable pumping mechanisms should be provided. One possibility for
this is electrophoretic movement of the fluids through electrodes
integrated into the support, or by reducing the volume in the
chambers of the support by appropriate introduction of force from
outside into the support (conventional pump).
[0144] 6.2.4 Starting Materials in the Synthesis Unit
[0145] In principle, the provision of the starting materials for
the support synthesis in storage containers offers the advantage of
multiplexing of finished base sequences and parallel channels,
which is why this design variant is advisable for (ultra)high
throughput screening and support manufacturers. The multiplexing
can take place at the interface to the support in which a specific
base sequence wets a different channel for each synthesis cycle. A
more technically elaborate but, where appropriate, more reliable
method is multiplexing in the equipment through an appropriate
valve system. Account must be taken here of cross-contamination,
which may arise through the use of different base sequences.
[0146] Another point which must be taken into account is the
collection and disposal of excess material at the exit from the
individual channels. It is conceivable in this connection both to
circulate (reuse the emerging material) and to dispose of the
emerging starting materials.
[0147] 6.3 Analyte Determination
[0148] Analysis of nucleic acid sequences takes place as with other
oligonucleotide arrays by hybridization of nucleic acids in the
sample material onto complementary strands among the immobilized
oligonucleotides.
[0149] As another possible use of the support, is to couple peptide
sequences in the channels, likewise in accordance with in situ
synthesis principles. Such peptides are capable of diverse and, in
some cases, highly specific binding reactions with peptides,
proteins and other substances, so that the range of potential
analytes can be considerably extended.
[0150] Synthesis in the support would make available for the first
time very highly parallel and, at the same time, cost-effective
peptide arrays for a large number of applications.
[0151] 6.3.1 Analytes
[0152] Examples of analytes are nucleic acids (DNA, RNA, in
specific cases also PNA). These nucleic acids can be obtained from
complete genomes, fragments thereof, chromosomes, plasmids or
synthetic sources (for example cDNA). In one embodiment, the sample
material may originate from the human genome.
[0153] Further examples of analytes are proteins, polypeptides and
peptides of all types, for example hormones, growth factors,
enzymes, tumor antigens, serum factors, antibodies, carbohydrates,
for example various sugars in foodstuffs or agricultural crops,
functional sugars, polymers and other organic molecules, for
example drugs of abuse, pharmaceuticals, metabolites, amino acids,
transmitters, pesticides, insecticides, paints, various toxins
etc.
[0154] 6.3.2 Variants for Binding to the Immobilized Interaction
Partner (Receptor)
[0155] The binding of the analyte to the receptor can in the case
of nucleic acids take place by hybridization of complementary
nucleic acids, for example longer molecules such as cDNA, synthetic
oligonucleotides, PNA, RNA. Peptides as receptors, for example
synthetic peptides or natural peptides, can bind to the analyte via
protein-protein or protein-nucleic acid interactions.
[0156] 6.3.3 Variants for Signal Generation
[0157] Two principles are preferably employed for signal
generation, namely: direct detection of an analyte which was
labeled beforehand or during the reaction (preferred method in
nucleic acid analysis by means of hybridization) and indirect
detection through competition of the analyte or the target sequence
with a labeled standard. The first variant is well established for
some applications, but tends to be rather unsuitable for
diagnostics for example of serum components, which is possible with
peptide arrays also in the support. The second variant is therefore
to be preferred for these applications, and it moreover allows in
principle, sample preparation by the user to be simpler.
[0158] Direct detection can take place by labeling the analytes
with a dye for absorption measurement, a fluorescent dye, labeling
the analytes with reporter enzyme, subsequent reaction (for example
chemo- or bioluminescence), selective labeling of the bound
analyte, for example in the case of nucleic acids by intercalating
(fluorescent) dyes, double strand-binding proteins or double
strand-binding antibodies or a secondary detection of the bound
analyte with a second component, for example in the case of PNA-DNA
hybrids by DNA-specific antibodies. Labeled standards which can be
used are enzyme-coupled standards (for example chemo- and
bioluminescence with alkaline phosphatase, peroxidase etc.) or
(fluorescence) dye-coupled standards. Protein standards can be
employed as fusion proteins with a reporter enzyme (see above) or
an autofluorescent protein (for example GFP), for example for
recombinant antibodies, protein hormones, growth factors etc.
[0159] 6.4 Provision of the Sample Material
[0160] There are likewise various design variants for the provision
of the sample material. The nature of the provision is irrelevant
to the actual detection because it is always necessary to provide a
sufficient amount of in liquid dissolved DNA fragments at the
interface for the desired investigation.
[0161] 6.4.1 External Sample Preparation
[0162] The sample preparation can take place either manually in the
laboratory, in a separate analysis system or in a preparation unit
integrated into the same system. The sample ready for detection is
then introduced into the support by means of manual or automatic
pipetting or comparable methods.
[0163] 6.4.2 Sample Preparation in the Same Support All in One
[0164] Precisely when multiplexing is used to reduce the production
times in the support synthesis it is possible to achieve identical
or even shorter times for the receptor synthesis than would be
necessary, for example, for DNA amplification of the sample by PCR.
This makes it worthwhile to integrate a PCR into the synthesis
system or even into the support for many applications.
[0165] Besides the time-consuming PCR, it is also possible to
integrate the preceding cell disruption, for example via readily
automatable methods such as ultrasound or high voltage, just like
the DNA isolation.
[0166] 6.5 Detection Unit
[0167] The reading of the light signals for the detection reactions
in the support array is to take place in a detection unit where the
excitation light source (fluorescence, luminescence or absorption
as optical detection) is arranged directly opposite to the CCD chip
for light signal measurement. The support array is located between
light source and detection chip (sandwich architecture). An
illumination matrix can be used as excitation light source. The
spatial arrangement of this unit may depend on requirements (for
example use of gravitation for flow processes in the chip). This
maximally compact architecture minimizes the paths traveled by the
light and thus also the intensity of light required. It is intended
to dispense with the use of an elaborate, light-consuming and
costly optical system which occupies much space, both on the
excitation side and on the detection side.
[0168] 6.5.1 Temperature During the Hybridization
[0169] The temperature control (at present typically 60.degree.
C.--most recent developments also now make hybridization possible
at 25.degree. C. with low-salt conditions) during the hybridization
can take place either by appropriate temperature elements in the
detection unit or by the excitation light source or the excitation
light per se. Temperature elements in the supports are likewise
possible.
[0170] 6.5.2 Excitation Light Source
[0171] Suitable light sources are, depending on the analyte markers
(detection method via absorption or fluorescence etc.), highly
parallel light from a lamp (white light), highly parallel light
from a flash tube, highly parallel monochromatic light, a
monochromatic streak of laser light, extensive illumination through
widening of the laser beam, a monochromatic laser beam or a
programmable light source matrix.
[0172] An appropriate optical grating or an appropriate optical
system can, where appropriate, be provided between excitation light
source and support array.
[0173] 6.5.3 CCD Camera Detection
[0174] The detection unit preferably consists of only one CCD chip.
These currently have about 2 000.times.3 000 pixels on an area of,
for example, 25.times.37 mm (Cannon). Arrangement of about 500
parallel channels with a diameter of about 20 .mu.m (every second
double pixel row) on such an area of 25.times.37 mm results in 750
measurement points (fields) in each channel if only every second
double pixel is used under the channel. This would provide 375 000
reaction regions on a single support, each reaction region covering
4 colored and 12 black and white pixels and having an area of
20.times.20 .mu.m. The light signals must be generated with maximum
density on the optical CCD chip so that faulty assignment of light
signals and measurement points with their specific base sequence,
and overlap of adjacent light signals, can be precluded. Otherwise,
serial detection of overlapping regions is possible, or fiber optic
elements are employed.
[0175] The resulting large number of measurements
(4.times.500.times.750=1- .5 million colored signals or 4.5 million
intensities between 0 and 4 096 digital values) which are available
(current CCD chip state of the art) form the basis permitting
extensive statistics in the analysis of the detected light signals.
The processing of the resulting quantities of data is possible
without difficulty through the development in efficiency with, at
the same time, fall in price of modern computer systems.
[0176] The detection of the detection reaction can provide both
qualitative and quantitative information, in particular which
capture molecules (position in the array) have found binding
partners (evaluation of the, for example, fluorescent label) and
how many capture molecules in a class have found a hybridization
partner.
[0177] It is possible where appropriate to provide an appropriate
optical grating or an appropriate optical system between the
support array and the CCD camera.
[0178] If detection with a CCD camera or a CCD chip does not
provide adequate signals, detection in the analytical system is
also possible by other, more sensitive sensors.
[0179] Of interest in connection with the present invention is the
use of an inspection unit as described in the German patent
application 198 39254.0. This inspection unit comprises an
electronically controllable light source matrix and a light sensor
matrix which is located opposite to and faces the light source
matrix, namely CCD image recorder.
[0180] It is conceivable in this connection that the user produces
his supports himself and uses them directly. He simply downloads
the required data (DNA sequences) from a CD-ROM or from the
Internet and produces in his illumination matrix-CCD unit his
individual DNA chip, then wets it with the sample and reads the
signals.
[0181] If, for example, every second pixel in this arrangement is
measured for the photoactivation, it is possible to use the pixels
in between, which lie in projection inside a channel, for a
permanent process control. Thus, for example, it is possible of
individual and dynamic following of the flowing in of a gas bubble
between two fluids in a channel. It would also be conceivable to
color the carrier fluids for G, A, C and T, so that the presence of
the correct oligos could be checked and a color change might signal
a cross-contamination. In the subsequent detection there could in
turn be site-specific and, if necessary, even color-specific light
excitation. This results in entirely novel possibilities for
detection methods currently not available as yet.
[0182] Using the inspection unit (illumination matrix-CCD unit), it
is possible to monitor the flow processes in the channels in a
support both during the production--i.e. in the oligo
synthesis--and during the analysis. For this purpose it is possible
to use, for example, cleansing gas bubbles between two fluids in
the channels or a coloring of the individual fluids.
[0183] It is possible to use an illumination matrix which generates
and transmits the necessary wavelength of, for example, 360-370 nm,
for photoinduced elimination of protective groups during the
synthesis of DNA oligos on or in the support.
[0184] Detection of the detection reaction in the support can
likewise take place in the inspection unit. If the detection is
achieved via fluorescent markers, it would be necessary, where
appropriate, to change the background illumination (automatically
possible). For this purpose it would be possible to use optical
filters or/and glass fiber elements (tapers). Where appropriate
novel detection methods are also used which is made possible only
by the extremely flexible, individual irradiation and detection of
the individual reaction region.
[0185] A temperature of about 55-65.degree. C. is required for
standard hybridization of DNA, RNA and PNA strands with one
another. In the simplest case, this temperature can be generated by
the energy emitted by the illumination matrix (waste heat and
wavelength). This would allow the arrangement to be made more
compact.
8. EXEMPLARY EMBODIMENTS
[0186] The synthesis of DNA molecules in channels can take place
with use of standard synthons, for example phosphoramidite building
blocks, with suitable protective groups, for example
dimethoxytrityl (DMT). A corresponding fluidic DNA synthesis can
take place starting from a linker coupled to the solid phase.
[0187] This format can be combined for the preferred embodiment of
the invention with a light-dependent control of the DNA synthesis.
For this purpose, protective groups which permit light-dependent
deprotection are known, so that the protective group, which is
usually linked on the 5' carbon atom of the synthon, is eliminated
by light of suitable wavelength. The synthesis of nucleic acids
with a length of 18 or more nucleotides is possible in capillaries
in this way.
[0188] The reaction products can be analyzed, for example by high
performance liquid chromatography (HPLC), by detaching the
synthesized DNA oligomer, as is possible on use of suitable
linkers. In this case it is possible to show the efficiency of the
capillary DNA synthesis via the proportion of full-length
products.
[0189] For light-dependent DNA synthesis, the reaction region on
the support is illuminated site- or/and time-specifically with a
suitable light source, for example with a mercury vapor lamp, laser
light (for example 373 nm nitrogen laser) or with a UV LED. Other
light sources which have sufficiently high-energy radiation are
likewise suitable too.
[0190] FIG. 1 shows a highly schematic plan view of a support
according to the invention.
[0191] FIG. 2 shows examples of channel arrangements in a support
according to the invention.
[0192] FIG. 3 shows a diagrammatic depiction of a support in an
inspection unit composed of programmable light source matrix and
CCD matrix.
[0193] FIG. 4 shows a diagrammatic depiction of an apparatus of the
invention for a light-assisted integrated synthesis and analysis
method and
[0194] FIG. 5 shows the structure from FIG. 4 for a fluidic
individualization of reaction regions.
[0195] FIG. 1 shows a transparent support in a plan view in a
highly schematic manner. The channels 1 which run parallel to one
another are evident, for example 500 channels with a length of 37
nm. T, G, A, C in FIG. 1 designate reservoirs for the individual
starting materials (bases). 3 designates the gas inlet. 5
identifies a valve. 7 identifies the sample input, and 9 designates
an entry for further synthetic chemicals and cleaning/washing
liquid.
[0196] FIG. 2 is a diagrammatic depiction of other examples of
alternative channel arrangements.
[0197] FIG. 3 shows the support of FIG. 1 in an inspection unit
composed of programmable light source matrix, for example an LCD
matrix, and a CCD detection matrix.
[0198] FIG. 4 depicts an apparatus of the invention with an
exchangeable support 40, the structure in principle depending on
whether the support is changed in each cycle or only when worn. In
the latter case there is cleaning and subsequent reuse of the same
channels. A programmable light source matrix 30 is depicted. Its
programmability can be integrated into the system component 20,
which consists of a calculator or a computer, so that only one
freely controllable light source matrix is necessary as component
30. This light source matrix 30 beams light of defined wavelength
and intensity onto any addressable sites of an at least
two-dimensional matrix which serves for highly parallel
illumination of the reaction regions in the support 40. Said
support 40 is irradiated individually by the light source matrix 30
with the computer-controlled light pattern consisting of energy
waves in all reaction regions. Fluids provided by the fluidics
module 60 are transported via the fluidic connection system 64 into
the support 40 and conveyed further in its microstructure, which is
not depicted in the drawing, in a suitable manner to the reaction
regions. The support 40 becomes an optofluidic microprocessor in
this way. The latter can be either changed after each use or
cleaned after each use and changed only for servicing purposes when
worn.
[0199] The entering light can be used, for example, for absorption
measurements, to activate photoreactions or to excite
fluorescence.
[0200] The light emerging from the support 40 or from the
optofluidic microprocessor can, for example, be the light from the
light source matrix 30 transmitted through the support. It can,
however, in this case also comprise light signals which are
generated in the individual reaction regions of the support 40 by,
for example, fluorescence or luminescence.
[0201] The detector matrix 50, which consists for example of a CCD
chip with or without optical system, is arranged in relation to a
light source matrix 30, with a support 40 being located in between,
so that the result is a triple matrix arrangement composed of light
matrix, support and detector matrix.
[0202] The fluidic module 60 serves to supply the reaction support
40 for example with starting materials, protective gases, chemicals
such as solvents etc., and sample material. The fluidic module 60
consists of tanks 61 which are emptied in a suitable manner by
pumps 62 and valves 63. The tanks can be exchanged or refilled
singly or in a cluster. Fluids which are required permanently, such
as, for example, protective gas, can also be supplied continuously
by means of lines (without tanks in the system). The fluidic waste
from the various methods can be collected either in tanks
integrated in the support 40 or in a waste system 65 or, in the
case of clusters, outside the individual system.
[0203] The system boundary 10 of the apparatus, which can be
employed as a single device or else in central or decentral
clusters, is likewise depicted. There is always information
technology linkage between these clusters. The systems located at a
site may also be supplied together, by manual operation or
automated components, with energy, fluids such as starting
materials, reaction chemicals, protective gases and sample
material, and with the required supports.
[0204] The system component 20 in the form of a computer or
calculator undertakes the control of the system. This includes the
control, on the basis of the calculation of the probe or receptor
sequences for the individual reaction regions, of the light source
matrix 30 and of the fluidic component 60. The data from the
detector matrix 50 are moreover collected and evaluated.
[0205] Each apparatus can thus communicate beyond its system
boundary 10 with other apparatuses or systems consisting in turn of
an apparatus of the invention or other computers or databases. This
can take place, for example, via lines, bus systems or via the
Internet. It is moreover possible for communication to take place
with central coordination by a master computer or as cluster of
equal-access systems. A data interface 21 to the system environment
is likewise provided.
[0206] FIG. 5 shows the structure from FIG. 4 for a fluidic
individualization of the reaction regions. A support 41 is depicted
once again. This is utilized individually under computer control by
the fluidic deprotection module 32. The fluids provided by the
fluidic module 60 are transported via the fluidic connection system
64 into the support and, in its microstructure which is not
depicted in the drawing, conveyed further in a suitable manner to
the reaction regions. This makes the support 41 into an optofluidic
microprocessor. The latter can be either changed after each use or
cleaned after each use and changed only for servicing purposes when
worn.
[0207] It is possible to feed light into this support for example
from above or/and from the side to excite fluorescence reactions
etc.
[0208] The light emerging from the support or the optofluidic
microprocessor can be generated, for example, by luminescence on
the reaction regions.
[0209] The fluidic deprotection module 32 is able to bring each
reaction region on the support 41 into contact individually with
fluids by use of at least one of the wetting components 33 (for
example nozzles, capillaries etc.). It is possible in this way to
activate, for example, local chemical and biochemical
reactions.
[0210] The fluidic module 31 serves to supply the fluidic
deprotection module 32 with starting materials or chemicals. The
fluidic module 31 has a comparable structure to the module 60 and
consists, depending on requirements, of tanks, lines, valves
etc.
[0211] The detector matrix 50, which consists for example of a CCD
chip with or without optical system, is arranged in relation to a
fluidic deprotection module 32, with a support 41 located in
between, in such a way that once again a triple matrix arrangement
is produced thereby.
[0212] The fluidic module 60 serves to supply the support 41 for
example with starting materials, protective gases, chemicals such
as solvents etc., and sample material. The fluidic module 60
consists of tanks 61 which are emptied in a suitable manner by
pumps 62 and valves 63. The tanks can be exchanged or refilled
singly or in a cluster. Fluids which are required permanently, such
as, for example, protective gas, can also be supplied continuously
by means of lines (without tanks in the system). The fluidic waste
from the various methods can be collected either in tanks
integrated in the support 41 or in a waste system 65 or, in the
case of clusters, outside the individual system.
[0213] The system boundary 10, which has already been explained, of
the apparatus and the system component 20 in the form of a computer
or calculator, which undertakes the control of the system, is
depicted once again. This includes the control of the fluidic
modules 31 and 60, and of the fluidic deprotection module 32, on
the basis of the calculation of the probe sequences for the
individual reaction regions. The data from the detector matrix 50
are moreover collected and evaluated.
[0214] Each apparatus can thus communicate beyond its system
boundary 10 with other apparatuses or systems consisting in turn of
an apparatus of the invention or other computers or databases. This
can take place, for example, via lines, bus systems or via the
Internet. It is moreover possible for communication to take place
with central coordination by a master computer or as cluster of
equal-access systems. A data interface 21 to the system environment
is likewise provided.
* * * * *